Tumor homing molecules, conjugates derived therefrom, and methods of using same

ABSTRACT

The present invention provides tumor homing molecules, which selectively home to a tumor. The invention also provides methods of using a tumor homing molecule to target an agent such as a drug to a selected tumor or to identify the target molecule expressed by the tumor. The invention also provides methods of targeting a tumor containing angiogenic vasculature by contacting the tumor with a molecule that specifically binds an α v -containing integrin. The invention further provides molecules that can selectively home to angiogenic vasculature. In addition, the invention provides a target molecule, which is specifically bound by a tumor homing molecule and is expressed by angiogenic vasculature. The invention also provides antibodies that bind to the target molecule and peptidomimetics that competitively inhibit binding of a ligand to the target molecule.

This application is a continuation of U.S. Ser. No. 10/375,992, filedFeb. 27, 2003, which is a continuation of U.S. Ser. No. 08/926,914,filed Sep. 10, 1997, which claims the benefit of priority of U.S.Provisional Application No. 60/060,947, which was converted from U.S.Ser. No. 08/710,067, filed Sep. 10, 1996, each of which the entirecontents is incorporated herein by reference.

This invention was made with government support under CA 42507, CA62042, CA74238-01 and Cancer Center Support Grant CA 30199 awarded bythe National Institutes of Health. The government has certain rights inthis invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of cancer biologyand drug delivery and, more specifically, to peptides that selectivelyhome to a tumor, particularly a malignant tumor, to compositionscomprising an agent such as a therapeutic agent conjugated to such tumorhoming molecules, and to methods of using such molecules to target anagent to a tumor.

2. Background Information

Continuous developments over the past quarter century have resulted insubstantial improvements in the ability of a physician to diagnose acancer in a patient. For example, antibody based assays such as that forprostate specific antigen now allow early diagnosis of cancers such asprostate cancer. More recently, methods of genetic screening arebecoming available to identify persons that may be particularlysusceptible to developing a cancer. Genetic screening methods are basedon the identification of one or more mutations in a gene that correlateswith the development of a cancer. For example, the identification ofgenes such as BRCA1 and BRCA2 allowed the further identification ofmutations in these genes that, in some cases, can correlate withsusceptibility to developing breast cancer.

Unfortunately, methods for treating cancer have not kept pace with thosefor diagnosing the disease. Thus, while the death rate from variouscancers has decreased due to the ability of a physician to detect thedisease at an earlier stage, the ability to treat patients presentingwith more advanced disease has advanced only minimally.

A major hurdle to advances in treating cancer is the relative lack ofagents that can selectively target the cancer, while sparing normaltissue. For example, radiation therapy and surgery, which generally arelocalized treatments, can cause substantial damage to normal tissue inthe treatment field, resulting in scarring and, in severe cases, loss offunction of the normal tissue. Chemotherapy, in comparison, whichgenerally is administered systemically, can cause substantial damage toorgans such as bone marrow, mucosae, skin and the small intestine, whichundergo rapid cell turnover and continuous cell division. As a result,undesirable side effects such as nausea, loss of hair and drop in bloodcell count occur as a result of systemically treating a cancer patientwith chemotherapeutic agents. Such undesirable side effects often limitthe amount of a treatment that can be administered. Thus, cancer remainsa leading cause of patient morbidity and death.

Efforts have been made to increase the target specificity of variousdrugs. For example, where a unique cell surface marker is expressed by apopulation of cells making up a tumor, an antibody can be raised againstthe unique marker and a drug can be linked to the antibody. Uponadministration of the drug/antibody complex to the patient, the bindingof the antibody to the marker results in the delivery of a relativelyhigh concentration of the drug to the tumor. Similar methods can be usedwhere a particular cancer cell or the supporting cell or matrixexpresses a unique cell surface receptor or a ligand for a particularreceptor. In these cases, the drug can be linked to the specific ligandor to the receptor, respectively, thus providing a means to deliver arelatively high concentration of the drug to the tumor.

Tumors are characterized, in part, by a relatively high level of activeangiogenesis, resulting in the continual formation of new blood vesselsto support the growing tumor. Such angiogenic blood vessels aredistinguishable from mature vasculature. One of the distinguishingfeatures of angiogenic vasculature is that unique endothelial cellsurface markers are expressed. Thus, the blood vessels in a tumorprovide a potential target for directing a chemotherapeutic agent to thetumor, thereby reducing the likelihood that the agent will killsensitive normal tissues. Furthermore, if agents that target theangiogenic blood vessels in a tumor can be identified, there is alikelihood that the agents can be useful against a variety of differenttypes of tumors, since it is the target molecules in the angiogenicvessels that are recognized by such agents and not receptors specificfor the tumor cells. However, the use of molecules that can bindspecifically to tumor vasculature and target a chemotherapeutic agent tothe tumor has not been demonstrated.

While linking a drug to a molecule that homes to a tumor can providesignificant advantages for treatment over the use of a drug, alone, useof this method is severely limited by the scarcity of useful cellsurface markers expressed in a tumor. Thus, a need exists to identifymolecules that can selectively home to a tumor, particularly to thevasculature supporting the tumor. The present invention satisfies thisneed and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention relates to molecules that selectively home totumors, generally to the vasculature supporting the tumor. For example,the invention provides tumor homing peptides that contain, for example,the motif asparagine-glycine-arginine (NGR) or glycineserine-leucine(GSL), or the α_(v)-containing integrin binding motif,arginine-glycine-aspartic acid (RGD).

The invention also relates to compositions comprising a tumor homingmolecule, such as a tumor homing peptide, linked to a moiety to producea tumor homing molecule/moiety conjugate. Such a moiety can be a drug,for example, a cancer therapeutic agent such as doxorubicin, taxol,cis-platinum, or the like, in which case the tumor homingmolecule/moiety conjugate provides a therapeutic reagent. A moietyconjugated to a tumor homing molecule also can be a detectable label,for example, a radionuclide or paramagnetic spin label, such that themolecule/moiety conjugate provides a diagnostic reagent.

The invention also relates to methods of targeting a moiety such as adrug to a tumor by contacting the tumor homing molecule/moiety conjugatewith the tumor. Thus, the invention provides methods of diagnosing ortreating a cancer in a subject by administering a composition comprisinga tumor homing molecule conjugated to a cancer therapeutic agent to thesubject. For example, administration of a composition comprising adoxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate to a mouse bearing atransplanted breast carcinoma substantially reduced the growth of thebreast cancer and the number of metastases and resulted in substantiallygreater survival as compared to tumor bearing mice treated withdoxorubicin, alone, or with doxorubicin conjugated to an unrelatedpeptide.

The invention further relates to methods of identifying a targetmolecule in a tumor by detecting selective binding of the targetmolecule to a tumor homing molecule. For example, a peptide thatselectively homes to a tumor can be attached to a solid matrix for usein affinity chromatography. A sample of the tumor can be obtained andpassed over the affinity matrix under conditions that allow specificbinding of the target molecule, which then can be collected andidentified using well known biochemical methods. Thus, the inventionalso provides a target molecule, which acts as a receptor for a tumorhoming molecule. Such a target molecule can be useful, for example, forraising an antibody specific for the target molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1V show the immunohistochemical staining of phage expressingthe NGR tumor homing peptide, CNGRCVSGCAGRC (SEQ ID NO: 3; “NGR phage”),in tumors and normal tissues following intravenous injection into nudemice bearing a human breast carcinoma or a human Kaposi's sarcoma.Samples were taken 4 min (FIGS. 1E, 1G, 1H and 1J) or 24 hr (FIGS. 1A to1D, 1F, 1I, and 1K to 1V) after administration of the phage. FIGS. 1A,1C, 1G and 1J are from mice receiving insertless phage (control phage)and FIGS. 1B, 1D, 1E, 1F, 1H, 1I and 1K to 1V are from mice receivingNGR phage. FIGS. 1A, 1B, 1E, 1F and 1G are breast tumor samples; FIGS.1C, 1D, 1H, 1I and 1J are Kaposi's sarcoma samples; FIG. 1K is brain;FIG. 1L is lymph node; FIG. 1M is kidney; FIG. 1N is pancreas; FIG. 10is uterus; FIG. 1P is mammary fat pad; FIG. 1Q is lung; FIG. 1R isintestine; FIG. 1S is skin; FIG. 1T is skeletal muscle; FIG. 1U is heartand FIG. 1V is urinary tract epithelium. Magnification: FIGS. 1A to 1D,40×; FIGS. 1E to 1V, 200×.

FIGS. 2A to 2F show the hematoxylin and eosin stained tumor samples fromrepresentative pairs of mice treated two times with 5 μg equivalent ofdoxorubicin (FIGS. 2A to 2C) or doxorubicin/CDCRGDCFC (SEQ ID NO: 1)conjugate (FIGS. 2D to 2F). Paired mice had size matched tumors at thetime treatment was initiated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to molecules that selectively home totumors. For example, the invention provides tumor homing peptides suchas the peptides CGRECPRLCQSSC (SEQ ID NO: 2) and CNGRCVSGCAGRC (SEQ IDNO: 3), which were identified based on their ability to home to a breastcarcinoma, and the peptide CLSGSLSC (SEQ ID NO: 4, which was identifiedbased on its ability to home to a melanoma. Such tumor homing peptideswere identified using in vivo panning (see U.S. Pat. No. 5,622,699,issued Apr. 22, 1997; Pasqualini and Ruoslahti, Nature 380:364-365(1996), each of which is incorporated herein by reference).

The disclosed tumor homing peptides were identified based on theirhoming to various particular tumors. For example, in vivo panning wasperformed using a mouse bearing a human breast carcinoma xenograft andpeptides that homed to the breast tumor were identified. However, asdisclosed herein, such tumor homing peptides generally homed to othertypes of tumors, including a mouse melanoma and a human Kaposi'ssarcoma. Thus, while the tumor homing peptide CNGRCVSGCAGRC (SEQ ID NO:3) was identified by its ability to home in vivo to a breast tumor, thispeptide also homed in vivo to a melanoma and to a Kaposi's sarcoma, butnot to nontumor tissues.

Similarly, the tumor homing peptide CLSGSLSC (SEQ ID NO: 4) wasidentified based on its homing to melanoma. However, further examinationof this peptide revealed that it also homed to a breast tumor and toKaposi's sarcoma. Immunohistological analysis revealed that such tumorhoming peptides initially are associated with the vasculature of thevarious tumors, although at later time the molecules are associated withtumor parenchymal cells. Thus, the general tumor homing ability of atumor homing molecule of the invention is due, at least in part, to theability of the tumor homing molecules to target angiogenic vasculatureassociated with a tumor. These results indicate that specific targetmolecules are expressed by the cells comprising the vasculature in atumor as compared to the cell surface molecule expressed by vasculaturein nontumor tissues. Using methods as disclosed herein, the artisanreadily can determine whether a tumor homing molecule homes generally tothe angiogenic vasculature associated with a tumor or homes specificallyto a particular type of tumor cell.

As used herein, the term “tumor” means a mass of cells that arecharacterized, at least in part, by containing angiogenic vasculature.The term “tumor” is used broadly to include the tumor parenchymal cellsas well as the supporting stroma, including the angiogenic blood vesselsthat infiltrate the tumor parenchymal cell mass. Although a tumorgenerally is a malignant tumor, i.e., a “cancer,” a tumor also can benonmalignant, provided that neovascularization is associated with thetumor. The term “normal” or “nontumor” tissue is used to refer to tissuethat is not a “tumor.” As disclosed herein, a tumor homing molecule canbe identified based on its ability to home a tumor, but not to acorresponding nontumor tissue.

As used herein, the term “corresponding,” when used in reference totumors or tissues or both, means that two or more tumors, or two or moretissues, or a tumor and a tissue are of the same histologic type. Theskilled artisan will recognize that the histologic type of a tissue is afunction of the cells comprising the tissue. Thus, the artisan willrecognize, for example, that a nontumor tissue corresponding to a breasttumor is normal breast tissue, whereas a nontumor tissue correspondingto a melanoma is skin, which contains melanocytes. Furthermore, forpurposes of the invention, it is recognized that a tumor homing moleculecan bind specifically to a target molecule expressed by the vasculaturein a tumor, which generally contains blood vessels undergoingneovascularization, in which case a tissue corresponding to the tumorwould comprise nontumor tissue containing blood vessels that are notundergoing active angiogenesis.

The term “corresponding” also is used herein in reference to theevolutionarily conserved nature of target molecules, which are expressedin a tumor, for example, in a mouse as compared to a human. Thus,reference to the corresponding target molecules in mouse tumorvasculature as compared, for example, to human vasculature, means targetmolecules having a similar function, particularly the ability tospecifically bind a tumor homing molecule.

Identified tumor homing molecules are useful, for example, for targetinga desired moiety such as a drug, a toxin or a detectable label, whichcan be linked to the molecule, to a tumor. Thus, the invention providestumor homing molecule/moiety conjugates, which are useful for targetingthe moiety to a tumor. Accordingly, the invention also provides methodsof targeting a moiety to a tumor and, therefore, methods of reducing theseverity of a tumor and of treating a subject having a cancer. (seeExample VII). In addition, a tumor homing molecule is useful foridentifying the target molecule, to which the homing molecule binds inthe tumor.

A tumor homing molecule can be identified by screening a library ofmolecules by in vivo panning as disclosed herein. As used herein, theterm “library” means a collection of molecules. A library can contain afew or a large number of different molecules, varying from about tenmolecules to several billion molecules or more. If desired, a moleculecan be linked to a tag, which can facilitate recovery or identificationof the molecule.

As used herein, the term “molecule” is used broadly to mean an organicchemical such as a drug; a nucleic acid molecule such as an RNA, a cDNAor an oligonucleotide; a peptide, including a variant or modifiedpeptide or peptide-like molecules, referred to herein aspeptidomimetics, which mimic the activity of a peptide; or a proteinsuch as an antibody or a growth factor receptor or a fragment thereofsuch as an Fv, Fd or Fab fragment of an antibody, which contains abinding domain. For convenience, the term “peptide” is used broadlyherein to mean peptides, proteins, fragments of proteins and the like. Amolecule also can be a non-naturally occurring molecule, which does notoccur in nature, but is produced as a result of in vitro methods or canbe a naturally occurring molecule such as a protein or fragment thereofexpressed from a cDNA library or a peptidomimetic.

As used herein, the term “peptidomimetic” is used broadly to mean apeptide-like molecule that has the binding activity of the tumor homingpeptide. With respect to the tumor homing peptides of the invention,peptidomimetics, which include chemically modified peptides,peptide-like molecules containing non-naturally occurring amino acids,peptoids and the like, have the binding activity of a tumor homingpeptide upon which the peptidomimetic is derived (see, for example,“Burger's Medicinal Chemistry and Drug Discovery” 5th ed., vols. 1 to 3(ed. M. E. Wolff; Wiley Interscience 1995), which is incorporated hereinby reference). Peptidomimetics provide various advantages over apeptide, including that a peptidomimetic can be stable during passagethrough the digestive tract and, therefore, useful for oraladministration.

Methods for identifying a peptidomimetic are well known in the art andinclude, for example, the screening of databases that contain librariesof potential peptidomimetics. For example, the Cambridge StructuralDatabase contains a collection of greater than 300,000 compounds thathave known crystal structures (Allen et al., Acta Crystallogr. SectionB, 35:2331 (1979)). This structural depository is continually updated asnew crystal structures are determined and can be screened for compoundshaving suitable shapes, for example, the same shape as a tumor homingmolecule, as well as potential geometrical and chemical complementarityto a target molecule bound by a tumor homing peptide. Where no crystalstructure of a tumor homing peptide or a target molecule, which bindsthe tumor homing molecule, is available, a structure can be generatedusing, for example, the program CONCORD (Rusinko et al., J. Chem. Inf.Comput. Sci. 29:251 (1989)). Another database, the Available ChemicalsDirectory (Molecular Design Limited, Information Systems; San LeandroCalif.), contains about 100,000 compounds that are commerciallyavailable and also can be searched to identify potential peptidomimeticsof a tumor homing molecule.

Methods for preparing libraries containing diverse populations ofvarious types of molecules such as peptides, peptoids andpeptidomimetics are well known in the art and various libraries arecommercially available (see, for example, Ecker and Crooke,Biotechnology 13:351-360 (1995), and Blondelle et al., Trends Anal.Chem. 14:83-92 (1995), and the references cited therein, each of whichis incorporated herein by reference; see, also, Goodman and Ro,Peptidomimetics for Drug Design, in “Burger's Medicinal Chemistry andDrug Discovery” Vol. 1 (ed. M. E. Wolff; John Wiley & Sons 1995), pages803-861, and Gordon et al., J. Med. Chem. 37:1385-1401 (1994), each ofwhich is incorporated herein by reference). Where a molecule is apeptide, protein or fragment thereof, the molecule can be produced invitro directly or can be expressed from a nucleic acid, which can beproduced in vitro. Methods of synthetic peptide and nucleic acidchemistry are well known in the art.

A library of molecules also can be produced, for example, byconstructing a cDNA expression library from mRNA collected from a cell,tissue, organ or organism of interest. Methods for producing suchlibraries are well known in the art (see, for example, Sambrook et al.,Molecular Cloning: A laboratory manual (Cold Spring Harbor LaboratoryPress 1989), which is incorporated herein by reference). Preferably, apeptide encoded by the cDNA is expressed on the surface of a cell or avirus containing the cDNA. For example, cDNA can be cloned into a phagevector such as fuse (Example I), wherein, upon expression, the encodedpeptide is expressed as a fusion protein on the surface of the phage.

In addition, a library of molecules can comprise a library of nucleicacid molecules, which can be DNA or RNA or an analog thereof. Nucleicacid molecules that bind, for example, to a cell surface receptor arewell known (see, for example, O'Connell et al., Proc. Natl. Acad. Sci.USA 93:5883-5887 (1996); Tuerk and Gold, Science 249:505-510 (1990);Gold et al., Ann. Rev. Biochem. 64:763-797 (1995), each of which isincorporated herein by reference). Thus, a library of nucleic acidmolecules can be administered to a subject having a tumor and tumorhoming molecules can be identified by in vivo panning. If desired, thenucleic acid molecules can be nucleic acid analogs that, for example,are less susceptible to attack by nucleases (see, for example, Jelineket al., Biochemistry 34:11363-11372 (1995); Latham et al., Nucl. AcidsRes. 22:2817-2822 (1994); Tam et al., Nucl. Acids Res. 22:977-986(1994); Reed et al., Cancer Res. 59:6565-6570 (1990), each of which isincorporated herein by reference).

As disclosed herein, in vivo panning for the purpose of identifying atumor homing molecule comprises administering a library to a subject,collecting a sample of a tumor and identifying a tumor homing molecule.The presence of a tumor homing molecule can be identified using variousmethods well known in the art. Generally, the presence of a tumor homingmolecule in a tumor is identified based on one or more characteristicscommon to the molecules present in the library, then the structure of aparticular tumor-homing molecule is identified. For example, a highlysensitive detection method such as mass spectrometry, either alone or incombination with a method such as gas chromatography, can be used toidentify tumor homing molecules in a tumor. Thus, where a librarycomprises diverse molecules based generally on the structure of anorganic molecule such as a drug, a tumor homing molecule can beidentified by determining the presence of a parent peak for theparticular molecule.

If desired, the tumor can be collected, then processed using a methodsuch as HPLC, which can provide a fraction enriched in molecules havinga defined range of molecular weights or polar or nonpolarcharacteristics or the like, depending, for example, on the generalcharacteristics of the molecules comprising the library. Conditions forHPLC will depend on the chemistry of the particular molecule and arewell known to those skilled in the art. Similarly, methods for bulkremoval of potentially interfering cellular materials such as DNA, RNA,proteins, lipids or carbohydrates are well known in the art; as aremethods for enriching a fraction containing an organic molecule using,for example, methods of selective extraction. Where a library comprisesa population of diverse organic chemical molecules, each linked to aspecific oligonucleotide tag, such that the specific molecule can beidentified by, determining the oligonucleotide sequence using polymerasechain reaction (PCR), genomic DNA can be removed from the sample of thecollected tumor in order to reduce the potential for background PCRreactions. In addition, a library can comprise a population of diversemolecules such as organic chemical molecules, each linked to a common,shared tag. Based on the presence and properties of the shared tag,molecules of the library that selectively home to a tumor can besubstantially isolated from a sample of the tumor. These and othermethods can be useful for enriching a sample of a collected tumor forthe particular tumor homing molecule, thereby removing potentiallycontaminating materials from the collected tumor sample and increasingthe sensitivity of detecting a molecule.

Evidence provided herein indicates that a sufficient number of tumorhoming molecules selectively homes to a tumor during in vivo panningsuch that the molecules readily can be identified. For example, variousindependent phage expressing the same peptide were identified in tumorsformed from implanted human breast cancer cells (Table 1), from mousemelanoma cells (Table 2) or from human Kaposi's sarcoma cells (Table 3).

Although a substantial fraction of the identified tumor homing moleculeshave the same structure, the peptide inserts of only a small number ofisolated phage were determined. It should be recognized, however, thathundreds of thousands to millions of phage expressing organ homingpeptides have been recovered following in vivo pannings for organ homingmolecules (see, for example, U.S. Pat. No. 5,622,699; Pasqualini andRuoslahti, supra, 1996). These results indicate that specific tumorhoming molecules will be present in substantial numbers in a tumorfollowing in vivo homing, thereby increasing the ease with which themolecules can be identified.

Ease of identification of a tumor homing molecule, particularly anuntagged molecule, depends on various factors, including the presence ofpotentially contaminating background cellular material. Thus, where thetumor homing molecule is an untagged peptide, a larger number must hometo the tumor in order to identify the specific peptides against thebackground of cellular protein. In contrast, a much smaller number of anuntagged organic chemical homing molecule such as a drug is identifiablebecause such molecules normally are absent from or present in only smallnumbers in the body. In such a case, a highly sensitive method such asmass spectrometry can be used to identify a tumor homing molecule. Theskilled artisan will recognize that the method of identifying a moleculewill depend, in part, on the chemistry of the particular molecule.

Where a tumor homing molecule is a nucleic acid or is tagged with anucleic acid, an assay such as PCR can be particularly useful foridentifying the presence of the molecule because, in principle, PCR candetect the presence of a single nucleic acid molecule (see, for example,Erlich, PCR Technology: Principles and Applications for DNAAmplification (Stockton Press 1989), which is incorporated herein byreference). Preliminary studies have demonstrated that, followingintravenous injection of 10 ng of an approximately 6000 base pairplasmid into a mouse and 2 minutes in the circulation, the plasmid wasdetectable by PCR in a sample of lung. These results indicate thatnucleic acids are sufficiently stable when administered into thecirculation such that in vivo panning can be used to identify nucleicacid molecules that selectively home to a tumor.

The molecules of a library can be tagged, which can facilitate recoveryor identification of the molecule. As used herein, the term “tag” meansa physical, chemical or biological moiety such as a plastic microbead,an oligonucleotide or a bacteriophage, respectively, that is linked to amolecule of the library. Methods for tagging a molecule are well knownin the art (Hermanson, Bioconjugate Techniques (Academic Press 1996),which is incorporated herein by reference).

A tag, which can be a shared tag or a specific tag, can be useful foridentifying the presence or structure of a tumor homing molecule of alibrary. As used herein, the term “shared tag” means a physical,chemical or biological moiety that is common to each molecule in alibrary. Biotin, for example, can be a shared tag that is linked to eachmolecule in a library. A shared tag can be useful to identify thepresence of a molecule of the library in a sample and also can be usefulto substantially isolate the molecules from a sample. For example, wherethe shared tag is biotin, the biotin-tagged molecules in a library canbe substantially isolated by binding to streptavidin or their presencecan be identified by binding with a labeled streptavidin. Where alibrary is a phage display library, the phage that express the peptidesare another example of a shared tag, since each peptide of the libraryis linked to a phage. In addition, a peptide such as the hemaglutininantigen can be a shared tag that is linked to each molecule in alibrary, thereby allowing the use of an antibody specific for thehemaglutinin antigen to substantially isolate molecules of the libraryfrom a sample of a selected tumor.

A shared tag also can be a nucleic acid sequence that can be useful toidentify the presence of molecules of the library in a sample or tosubstantially isolate molecules of a library from a sample. For example,each of the molecules of a library can be linked to the same selectednucleotide sequence, which constitutes the shared tag. An affinitycolumn containing a nucleotide sequence that is complementary to theshared tag then can be used to hybridize molecules of the librarycontaining the shared tag, thus substantially isolating the moleculesfrom a tumor sample. A nucleotide sequence complementary to a portion ofthe shared nucleotide sequence tag also can be used as a PCR primer suchthat the presence of molecules containing the shared tag can beidentified in a sample by PCR.

A tag also can be a specific tag. As used herein, the term “specifictag” means a physical, chemical or biological tag that is linked to aparticular molecule in a library and is unique for that particularmolecule. A specific tag is particularly useful if it is readilyidentifiable. A nucleotide sequence that is unique for a particularmolecule of a library is an example of a specific tag. For example, themethod of synthesizing peptides tagged with a unique nucleotide sequenceprovides a library of molecules, each containing a specific tag, suchthat upon determining the nucleotide sequence, the identity of thepeptide is known (see Brenner and Lerner, Proc. Natl. Acad. Sci., USA89:5381-5383 (1992), which is incorporated herein by reference). The useof a nucleotide sequence as a specific tag for a peptide or other typeof molecule provides a simple means to identify the presence of themolecule in a sample because an extremely sensitive method such as PCRcan be used to determine the nucleotide sequence of the specific tag,thereby identifying the sequence of the molecule linked thereto.Similarly, the nucleic acid sequence encoding a peptide expressed on aphage is another example of a specific tag, since sequencing of thespecific tag identifies the amino acid sequence of the expressedpeptide.

The presence of a shared tag or a specific tag can provide a means toidentify or recover a tumor homing molecule of the invention followingin vivo panning. In addition, the combination of a shared tag andspecific tag can be particularly useful for identifying a tumor homingmolecule. For example, a library of peptides can be prepared such thateach is linked to a specific nucleotide sequence tag (see, for example,Brenner and Lerner, supra, 1992), wherein each specific nucleotidesequence tag has incorporated therein a shared tag such as biotin. Uponhoming to a tumor, the particular tumor homing peptides can besubstantially isolated from a sample of the tumor based on the sharedtag and the specific peptides can be identified, for example, by PCR ofthe specific tag (see Erlich, supra, 1989).

A tag also can serve as a support. As used herein, the term “support”means a tag having a defined surface to which a molecule can beattached. In general, a tag useful as a support is a shared tag. Forexample, a support can be a biological tag such as a virus or virus-likeparticle such as a bacteriophage (“phage”); a bacterium such as E. coli;or a eukaryotic cell such as a yeast, insect or mammalian cell; or canbe a physical tag such as a liposome or a microbead, which can becomposed of a plastic, agarose, gelatin or other biological or inertmaterial. If desired, a shared tag useful as a support can have linkedthereto a specific tag. Thus, the phage display libraries used in theexemplified methods can be considered to consist of the phage, which isa shared tag that also is a support, and the nucleic acid sequenceencoding the expressed peptide, the nucleic acid sequence being aspecific tag.

In general, a support should have a diameter less than about 10 μm toabout 50 μm in its shortest dimension, such that the support can passrelatively unhindered through the capillary beds present in the subjectand not occlude circulation. In addition, a support can be nontoxic, sothat it does not perturb the normal expression of cell surface moleculesor normal physiology of the subject, and biodegradable, particularlywhere the subject used for in vivo panning is not sacrificed to collecta selected tumor.

Where a molecule is linked to a support, the tagged molecule comprisesthe molecule attached to the surface of the support, such that the partof the molecule suspected of being able to interact with a targetmolecule in a cell in the subject is positioned so as to be able toparticipate in the interaction. For example, where the tumor homingmolecule is suspected of being a ligand for a growth factor receptor,the binding portion of the molecule attached to a support is positionedso it can interact with the growth factor receptor on a cell in thetumor. If desired, an appropriate spacer molecule can be positionedbetween the molecule and the support such that the ability of thepotential tumor homing molecule to interact with the target molecule isnot hindered. A spacer molecule also can contain a reactive group, whichprovides a convenient and efficient means of linking a molecule to asupport and, if desired, can contain a tag, which can facilitaterecovery or identification of the molecule (see Hermanson, supra, 1996).

As exemplified herein, a peptide suspected of being able to home to aselected tumor such as a breast carcinoma or a melanoma was expressed asthe N-terminus of a fusion protein, wherein the C-terminus consisted ofa phage coat protein. Upon expression of the fusion protein, theC-terminal coat protein linked the fusion protein to the surface of aphage such that the N-terminal peptide was in a position to interactwith a target molecule in the tumor. Thus, a molecule having a sharedtag was formed by the linking of a peptide to a phage, wherein the phageprovided a biological support, the peptide molecule was linked as afusion protein, the phage-encoded portion of the fusion protein acted asa spacer molecule, and the nucleic acid encoding the peptide provided aspecific tag allowing identification of a tumor homing peptide.

As used herein, the term “in vivo panning,” when used in reference tothe identification of a tumor homing molecule, means a method ofscreening a library by administering the library to a subject andidentifying a molecule that selectively homes to a tumor in the subject(see U.S. Pat. No. 5,622,699). The term “administering to a subject”,when used in referring to a library of molecules or a portion of such alibrary, is used in its broadest sense to mean that the library isdelivered to a tumor in the subject, which, generally, is a vertebrate,particularly a mammal such as a human.

A library can be administered to a subject, for example, by injectingthe library into the circulation of the subject such that the moleculespass through the tumor; after an appropriate period of time, circulationis terminated by sacrificing the subject or by removing a sample of thetumor (Example I; see, also, U.S. Pat. No. 5,622,699; Pasqualini andRuoslahti, supra, 1996). Alternatively, a cannula can be inserted into ablood vessel in the subject, such that the library is administered byperfusion for an appropriate period of time, after which the library canbe removed from the circulation through the cannula or the subject canbe sacrificed to collect the tumor, or the tumor can be sampled, toterminate circulation. Similarly, a library can be shunted through oneor a few organs, including the tumor, by cannulation of the appropriateblood vessels in the subject. It is recognized that a library also canbe administered to an isolated perfused tumor. Such panning in anisolated perfused tumor can be useful to identify molecules that bind tothe tumor and, if desired, can be used as an initial screening of alibrary.

The use of in vivo panning to identify tumor homing molecules isexemplified herein by screening a phage peptide display library intumor-bearing mice and identifying specific peptides that selectivelyhomed to a breast tumor or to a melanoma tumor (Example I). However,phage libraries that display protein receptor molecules, including, forexample, an antibody or an antigen binding fragment of an antibody suchan Fv, Fd or Fab fragment; a hormone receptor such as a growth factorreceptor; or a cell adhesion receptor such as an integrin or a selectinalso can be used to practice the invention. Variants of such moleculescan be constructed using well known methods such as random,site-directed or codon based mutagenesis (see Huse, U.S. Pat. No.5,264,563, issued Nov. 23, 1993, which is incorporated herein byreference) and, if desired, peptides can be chemically modifiedfollowing expression of the phage but prior to administration to thesubject. Thus, various types of phage display libraries can be screenedby in vivo panning.

Phage display technology provides a means for expressing a diversepopulation of random or selectively randomized peptides. Various methodsof phage display and methods for producing diverse populations ofpeptides are well known in the art. For example, Ladner et al. (U.S.Pat. No. 5,223,409, issued Jun. 29, 1993, which is incorporated hereinby reference) describe methods for preparing diverse populations ofbinding domains on the surface of a phage. In particular, Ladner et al.describe phage vectors useful for producing a phage display library, aswell as methods for selecting potential binding domains and producingrandomly or selectively mutated binding domains.

Similarly, Smith and Scott (Meth. Enzymol. 217:228-257 (1993); see,also, Scott and Smith, Science 249: 386-390 (1990), each of which isincorporated herein by reference) describe methods of producing phagepeptide display libraries, including vectors and methods of diversifyingthe population of peptides that are expressed (see, also, Huse, WO91/07141 and WO 91/07149, each of which is incorporated herein byreference; see, also, Example I). Phage display technology can beparticularly powerful when used, for example, with a codon basedmutagenesis method, which can be used to produce random peptides orrandomly or desirably biased peptides (Huse, U.S. Pat. No. 5,264,563,supra, 1993). These or other well known methods can be used to produce aphage display library, which can be subjected to the in vivo panningmethod of the invention in order to identify a peptide that homes to atumor.

In addition to screening a phage display library, in vivo panning can beused to screen various other types of libraries, including, for example,an RNA or DNA library or a chemical library. If desired, the tumorhoming molecule can be tagged, which can facilitate recovery of themolecule from the tumor or identification of the molecule in the tumor.For example, where a library of organic molecules, each containing ashared tag, is screened, the tag can be a moiety such as biotin, whichcan be linked directly to the molecule or can be linked to a supportcontaining the molecules. Biotin provides a shared tag useful forrecovering the molecule from a selected tumor sample using an avidin orstreptavidin affinity matrix. In addition, a molecule or a supportcontaining a molecule can be linked to a hapten such as4-ethoxy-methylene-2-phenyl-2-oxazoline-5-one (phOx), which can be boundby an anti-phOx antibody linked to a magnetic bead as a means to recoverthe molecule. Methods for purifying biotin or phOx labeled conjugatesare known in the art and the materials for performing these proceduresare commercially available (e.g., Invitrogen, La Jolla Calif.; andPromega Corp., Madison Wis.). In the case where a phage library isscreened, the phage can be recovered using methods as disclosed inExample I.

In vivo panning provides a method for directly identifying moleculesthat can selectively home to a tumor. As used herein, the term “home” or“selectively home” means that a particular molecule binds relativelyspecifically to a target molecule present in the tumor followingadministration to a subject. In general, selective homing ischaracterized, in part, by. detecting at least a two-fold (2×) greaterspecific binding of the molecule to the tumor as compared to a controlorgan or tissue.

It should be recognized that, in some cases, a molecule can localizenonspecifically to an organ or tissue containing a tumor. For example,in vivo panning of a phage display library can result in high backgroundin organs such as liver and spleen, which contain a marked component ofthe reticuloendothelial system (RES). Thus, where a tumor is present,for example, in the liver, nonspecific binding of molecules due touptake by the RES can make identifying a tumor homing molecule moredifficult.

Selective homing can be distinguished from nonspecific binding, however,by detecting differences in the abilities of different individual phageto home to a tumor. For example, selective homing can be identified bycombining a putative tumor homing molecule such as a peptide expressedon a phage with a large excess of non-infective phage or with about afive-fold excess of phage expressing unselected peptides, injecting themixture into a subject and collecting a sample of the tumor. In thelatter case, for example, provided the number of injected phageexpressing tumor homing peptide is sufficiently low so as to benonsaturating for the target molecule, a determination that greater thanabout 20% of the phage in the tumor express the putative tumor homingmolecule is demonstrative evidence that the peptide expressed by thephage is a specific tumor homing molecule. In addition, nonspecificlocalization can be distinguished from selective homing by performingcompetition experiments using, for example, phage expressing a putativetumor homing peptide in combination with an excess amount of the “free”peptide (Example IV).

In addition, various methods can be used to prevent nonspecific bindingof a molecule to an organ containing a component of the RES. Forexample, a molecule that homes selectively to a tumor present in anorgan containing a component of the RES can be obtained by firstblocking the RES using, for example, polystyrene latex particles ordextran sulfate (see Kalin et al., Nucl. Med. Biol. 20:171-174 (1993);Illum et al., J. Pharm. Sci. 75:16-22 (1986); Takeya et al., J. Gen.Microbiol. 100:373-379 (1977), each of which is .incorporated herein byreference), then administering the library to the subject. For example,pre-administration of dextran sulfate 500 or polystyrene microspheresprior to administration of a test substance has been used to blocknonspecific uptake of the test substance by Kupffer cells, which are theRES component of the liver (Illum et al., supra, 1986). Similarly,nonspecific uptake of agents by the RES has been blocked using carbonparticles or silica (Takeya et al., supra, 1977) or a gelatine colloid(Kalin et al., supra, 1993). Thus, various agents useful for blockingnonspecific uptake by the RES are known and routinely used.

Nonspecific binding of phage to RES or to other sites also can beprevented by coinjecting, for example, mice with a specific phagedisplay library together with the same phage made noninfective (Smith etal., supra, 1990, 1993). In addition, a peptide that homes to tumor inan organ containing an RES component can be identified by preparing aphage display library using phage that exhibit low background binding tothe particular organ. For example, Merrill et al. (Proc. Natl. Acad.Sci. USA 93:3188-3192 (1996), which is incorporated herein by reference)selected lambda-type phage that are not taken up by the RES and, as aresult, remain in the circulation for a prolonged period of time. Afilamentous phage variant, for example, can be selected using similarmethods.

Selective homing can be demonstrated by determining the specificity of atumor homing molecule for the tumor as compared to a control organ ortissue. Selective homing also can be demonstrated by showing thatmolecules that home to a tumor, as identified by one round of in vivopanning, are enriched for tumor homing molecules in a subsequent roundof in vivo panning. For example, phage expressing peptides thatselectively home to a melanoma tumor were isolated by in vivo panning,then were subjected to additional rounds of in vivo panning. Following asecond round of screening, phage recovered from the tumor showed a3-fold enrichment in homing to the tumor as compared to brain. Phagerecovered from the tumor after a third round of screening showed anaverage of 10-fold enrichment in homing to the tumor as compared tobrain. Selective homing also can be demonstrated by showing thatmolecules that home to a selected tumor, as identified by one round ofin vivo panning, are enriched for tumor homing molecules in a subsequentround of in vivo panning.

Tumor homing molecules can be identified by in vivo panning using, forexample, a mouse containing a transplanted tumor. Such a transplantedtumor can be, for example, a human tumor that is transplanted intoimmunodeficient mice such as nude mice or a murine tumor that ismaintained by passage in tissue culture or in mice. Due to the conservednature of cellular receptors and of ligands that bind a particularreceptor, it is expected that angiogenic vasculature and histologicallysimilar tumor cells in various species can share common cell surfacemarkers useful as target molecules for a tumor homing molecule. Thus,the skilled artisan would recognize that a tumor homing moleculeidentified using, for example, in vivo panning in a mouse having amurine tumor of a defined histological type such as a melanoma alsowould bind to the corresponding target molecule in a tumor in a human orother species. Similarly, tumors growing in experimental animals requireassociated neovascularization, just as that required for a tumor growingin a human or other species. Thus, a tumor homing molecule that binds atarget molecule present in the vasculature in a tumor grown in a mouselikely also can bind to the corresponding target molecule in thevasculature of a tumor in a human or other mammalian subject. Thegeneral ability of a tumor homing molecule identified, for example, byhoming to a human breast tumor, also to home to a human Kaposi's sarcomaor to a mouse melanoma indicates that the target molecules are shared bymany tumors. Indeed, the results disclosed herein demonstrate that thetarget molecules are expressed in the neovasculature, which is hosttissue (see Examples IV and VII).

A tumor homing molecule identified using in vivo panning in anexperimental animal such as a mouse readily can be examined for theability to bind to a corresponding tumor in a human patient bydemonstrating, for example, that the molecule also can bind specificallyto a sample of the tumor obtained from the patient. For example, theCDCRGDCFC (SEQ ID NO: 1) phage and NGR peptides have been shown to bindto blood vessels in microscopic sections of human tumors, whereas littleor no binding occurs in the blood vessels of nontumor tissues. Thus,routine methods can be used to confirm that a tumor homing moleculeidentified using in vivo panning in an experimental animal also can bindthe target molecule in a human tumor.

The steps of administering the library to the subject, collecting aselected tumor and identifying the molecules that home to the tumor,comprise a single round of in vivo panning. Although not required, oneor more additional rounds of in vivo panning generally are performed.Where an additional round of in vivo panning is performed, the moleculesrecovered from the tumor in the previous round are administered to asubject, which can be the same subject used in the previous round, whereonly a part of the tumor was collected.

By performing a second round of in vivo panning, the relative bindingselectivity of the molecules recovered from the first round can bedetermined by administering the identified molecules to a subject,collecting the tumor, and determining whether more phage is recoveredfrom the tumor following the second round of screening as compared tothose recovered following the first round. Although not required, acontrol organ or tissue also can be collected and the moleculesrecovered from the tumor can be compared with those recovered from thecontrol organ. Ideally, no molecules are recovered from a control organor tissue following a second or subsequent round of in vivo panning.Generally, however, a proportion of the molecules also will be presentin a control organ or tissue. In this case, the ratio of molecules inthe selected tumor as compared to the control organ (selected:control)can be determined. For example, phage that homed to melanoma following afirst round of in vivo panning demonstrated a 3× enrichment in homing tothe selected tumor as compared to the control organ, brain, followingtwo additional rounds of panning (Example V).

Additional rounds of in vivo panning can be used to determine whether aparticular molecule homes only to the selected tumor or can recognize atarget on the tumor that also is expressed in one or more normal organsor tissues in a subject or is sufficiently similar to the targetmolecule on the tumor. It is unlikely that a tumor homing molecule alsowill home to a corresponding normal tissue because the method of in vivopanning selects only those-molecules that home to the tumor, which isselected. Where a tumor homing molecule also directs homing to one ormore normal organs or tissues in addition to the tumor, the organs ortissues are considered to constitute a family of selected organs ortissues. Using the method of in vivo panning, molecules that home toonly the selected tumor can be distinguished from molecules that alsohome to one or more selected organs or tissues. Such identification isexpedited by collecting various organs or tissues during subsequentrounds of in vivo panning.

The term “control organ or tissue” is used to mean an organ or tissueother than the tumor for which the identification of a tumor homingmolecule is desired. A control organ or tissue is characterized in thata tumor homing molecule does not selectively home to the control organ.A control organ or tissue can be collected, for example, to identifynonspecific binding of the molecule or to determine the selectivity ofhoming of the molecule. In addition, nonspecific binding can beidentified by administering, for example, a control molecule, which isknown not to home to a tumor but is chemically similar to a potentialtumor homing molecule. Alternatively, were the administered moleculesare linked to a support, administration of the supports, alone, also canbe used to identify nonspecific binding. For example, a phage thatexpresses the gene III protein, alone, but that does not contain apeptide fusion protein, can be studied by in vivo panning to determinethe level of nonspecific binding of the phage support.

As disclosed herein, specific homing of a tumor homing molecule readilycan be identified by examining the selected tumor tissue as compared toa corresponding nontumor tissue, as well as to control organs ortissues. For example, immunohistological analysis can be performed on atumor tissue and corresponding nontumor tissue using an antibodyspecific for a phage used to display tumor homing peptides (see ExampleIV). Alternatively, an antibody can be used that is specific for ashared tag that expressed with the peptide, for example, a FLAG epitopeor the like, such detection systems being commercially available.

In general, a library of molecules, which contains a diverse populationof random or selectively randomized molecules of interest, is prepared,then administered to a subject. At a selected time after administration,the subject is sacrificed and the tumor is collected such that themolecules present in the tumor can be identified (see Example I). Ifdesired, one or more control organs or tissues or a part of a controlorgan or tissue can be sampled. For example, mice bearing a breast tumoror a melanoma tumor were injected with a phage peptide display library,then, after about 1 to 5 minutes, the mice were anesthetized, eitherfrozen in liquid nitrogen or, preferably, are perfused through the heartto terminate circulation of the phage, the tumor and one or more controlorgans were collected from each, phage present in the tumor and thecontrol organs were recovered and peptides that selectively homed to therespective tumors were identified (see Examples I, II and V).

In the examples provided, the animals were sacrificed to collect theselected tumor and control organ or tissue. It should be recognized,however, that only a part of a tumor need be collected to recover asupport containing a molecule that homes to that tumor and, similarly,only part of an organ or tissue need be collected as a control. Thus, apart of a tumor, for example, can be collected by biopsy, such that amolecule such as a peptide expressed by a phage can be administered tothe same subject a second time or more, as desired. Where the moleculethat is to be administered a second time to the same subject is taggedor linked, for example, to a support, the tag or support should benontoxic and biodegradable, so as not to interfere with subsequentrounds of screening.

In vitro screening of phage libraries previously has been used toidentify peptides that bind to antibodies or to cell surface receptors(Smith and Scott, supra, 1993). For example, in vitro screening of phagepeptide display libraries has been used to identify novel peptides thatspecifically bound to integrin adhesion receptors (Koivunen et al., J.Cell Biol. 124:373-380 (1994a), which is incorporated herein byreference) and to the human urokinase receptor (Goodson et al., Proc.Natl. Acad. Sci. USA 91:7129-7133 (1994)). However, such in vitrostudies provide no insight as to whether a peptide that can specificallybind to a selected receptor in vitro also will bind the receptor in vivoor whether the binding peptide or the receptor are unique to a specificorgan in the body. Furthermore, the in vitro methods are performed usingdefined, well-characterized target molecules in an artificial system.For example, Goodson et al. (supra, 1994) utilized cells expressing arecombinant urokinase receptor. However, such in vitro methods arelimited in that they require prior knowledge of the target molecule andyield little if any information regarding in vivo utility.

In vitro panning against cells in culture also has been used to identifymolecules that can specifically bind to a receptor expressed by thecells (Barry et al., Nature Med. 2:299-305 (1996), which is incorporatedherein by reference). However, the cell surface molecules that areexpressed by a cell in vivo often change when the cell is grown inculture. Thus, in vitro panning methods using cells in culture also arelimited, in that there is no guarantee a molecule that is identified dueto its binding to a cell in culture will have the same binding abilityin vivo. Furthermore, it is not possible using in vitro panning todistinguish molecules that home only to the tumor cells used in thescreening, but not to other cell types.

In contrast, in vivo panning requires no prior knowledge or availabilityof a target molecule and identifies molecules that bind to cell surfacetarget molecules that are expressed in vivo. Also, since the“nontargeted” tissues are present during the screening, the probabilityof isolating tumor homing molecules that lack specificity of homing isgreatly reduced. Furthermore, in obtaining tumor homing molecules by invivo panning, any molecules that may be particularly susceptible todegradation in the circulation in vivo due, for example, to a metabolicactivity, are not recovered. Thus, in vivo panning provides significantadvantages over previous methods by identifying molecules thatselectively home in vivo and the target molecule present in a tumor.

Although mechanisms by which the disclosed method of in vivo panningworks have not been fully defined, one possibility is that a moleculesuch as a peptide expressed on a phage recognizes and binds to a targetmolecule present on endothelial cells lining the blood vessels in atumor. Evidence indicates, for example, that the vascular tissues invarious organs differ from one another and that such differences can beinvolved in regulating cellular trafficking in the body. For example,lymphocytes home to lymph nodes or other lymphoid tissues due, in part,to the expression of specific “address” molecules by the endothelialcells in those tissues (Salmi et al., Proc. Natl. Acad. Sci., USA89:11436-11440 (1992); Springer, Cell 76:301-314 (1994)). Similarly,various leukocytes can recognize sites of inflammation due, in part, tothe expression of endothelial cell markers induced by inflammatorysignals (see Butcher and Picker, Science 272:60-66 (1996); Springer,supra, 1994). Thus; endothelial cell markers provide a potential targetfor directing, for example, a drug, which can be linked to a tumorhoming molecule, to a tumor in a subject.

In some cases, the metastasis of cancer cells to specific organs alsocan be due to recognition by the tumor cell of an organ specific marker,including organ specific endothelial cell markers (Fidler and Hart,Science 217:998-1003 (1982)). The pattern of metastasis of many cancerscan be explained by assuming that circulating tumor cells arepreferentially trapped in the first vascular bed encountered. Thus, thelungs and the liver are the most frequent sites of cancer metastasis:However, some cancers show patterns of metastasis that are not explainedby circulatory routing. Metastasis of such cancers may be due to thepresence of selectively expressed address molecules such as endothelialcell surface molecules expressed in the organ to which the cancermetastasizes (see Goetz et al., Int. J. Cancer 65:192-199 (1996); Zhu etal., Proc. Natl. Acad. Sci. USA 88:9568-9572 (1991); Pauli et al.,Cancer Metast. Rev. 9:175-189 (1990); Nicolson, Biochim. Biophys. Acta948:175-224 (1988)). The identification of molecules that bind to suchorgan-specific endothelial cell markers can provide a means to preventtumor cell metastasis to the particular organ.

The vasculature within a tumor generally undergoes active angiogenesis,resulting in the continual formation of new blood vessels to support thegrowing tumor. Such angiogenic blood vessels are distinguishable frommature vasculature in that angiogenic vasculature expresses uniqueendothelial cell surface markers, including the α_(v)β₃ integrin(Brooks, Cell 79:1157-1164 (1994); WO 95/14714, Int. Filing Date Nov.22, 1994) and receptors for angiogenic growth factors (Mustonen andAlitalo, J. Cell Biol. 129:895-898 (1995); Lappi, Semin. Cancer Biol.6:279-288 (1995)). Moreover, tumor vasculature is histologicallydistinguishable from blood vessel in general in that tumor vasculatureis fenestrated (Folkman, Nature Med. 1:27-31 (1995); Rak et al.,Anticancer Drugs 6:3-18 (1995)). Thus, the unique characteristics-oftumor vasculature make it a particularly attractive target fordetermining whether a molecule that homes specifically to a tumor can beidentified by in vivo panning. Such a tumor homing molecule can beuseful for directing an agent such as a chemotherapeutic drug to atumor, while reducing the likelihood the agent will have a toxic effecton normal, healthy organs or tissues (Example VII). Moreover, a moleculethat homes selectively to tumor vasculature also may have use intargeting other types of neovasculature such as that present ininflammatory, regenerating or wounded tissues.

Using in vivo panning to a breast carcinoma, a melanoma and a Kaposi'ssarcoma, phage expressing various peptides that selectively homed totumors were identified (see Tables 1, 2 and 3, respectively). Due to thelarge size of the phage (900-1000 nm) and the short time the phage wereallowed to circulate (3 to 5 min), it is unlikely that a substantialnumber of phage would have exited the circulatory system, particularlyin the brain and kidney. Tissue staining studies indicated that thetumor homing molecules that were identified primarily homed to and boundendothelial cell surface markers, which likely are expressed in anorgan-specific manner. These results indicate that in vivo panning canbe used to identify and analyze endothelial cell specificities. Such ananalysis is not possible using endothelial cells in culture because thecultured cells tend to lose their tissue-specific differences (Pauli andLee, Lab. Invest. 58:379-387 (1988)).

Although the conditions under which the in vivo pannings were performedidentified tumor homing peptides that generally bind to endothelial cellmarkers, the specific presence of phage expressing tumor homing peptidesalso was observed in tumor parenchyma, particularly at later times afteradministration of the peptides (Example IV). These results demonstratethat phage expressing peptides can pass through the blood vessels in thetumor, possibly, due to the fenestrated nature of the blood vessels, andindicate that the in vivo panning method can be useful for identifyingtarget molecules expressed by tumor cells, as well as target moleculesexpressed by endothelial cells.

Phage peptide display libraries were constructed essentially asdescribed Smith and Scott (supra, 1993; see, also, Koivunen et al.,Biotechnology 13:265-270 (1995); Koivunen et al., Meth. Enzymol.245:346-369 (1994b), each of which is incorporated herein by reference).Oligonucleotides encoding peptides having substantially random aminoacid sequences were synthesized based on an “NNK” codon, wherein “N” isA, T, C or G and “K” is G or T. “NNK” encodes 32 triplets, which encodethe twenty amino acids and an amber STOP codon (Scott and Smith, supra,1990). In some libraries, at least one codon encoding cysteine also wasincluded in each oligonucleotide so that cyclic peptides could be formedthrough disulfide linkages (Example I). The oligonucleotides wereinserted in frame with the sequence encoding the gene III protein (gIII)in the vector fuse 5 such that a peptide-gIII fusion protein can beexpressed. Following expression, the fusion protein is expressed on thesurface of the phage containing the vector (Koivunen et al., supra,1994b; Smith and Scott, supra, 1993).

Following in vivo panning, the phage isolated based on their ability toselectively home to human breast carcinoma, mouse melanoma or humanKaposi's sarcoma tumors displayed only a few different peptide sequences(see Tables 1, 2 and 3, respectively). One of the screenings revealedpeptide sequences that contained the arginine-glycine-aspartic acid(RGD) integrin recognition sequence (Ruoslahti, Ann. Rev. Cell Devel.Biol. 12:697 (1996)) in the context of a peptide previously demonstratedto bind selectively to aa-containing integrins (Koivunen et al., supra,1995; WO 95/14714). The sequences of most of the remaining tumor homingpeptides did not reveal any significant similarities with known ligandsfor endothelial cell receptors. However, one of the tumor homingpeptides contained the asparagine-glycine-arginine (NGR) motif, which isa weak integrin binding motif similar to the motifs present inintegrin-binding peptides (Ruoslahti et al., U.S. Pat. No. 5,536,814,issued Jul. 16, 1996, which is incorporated herein by reference; see,also, Koivunen et al., supra, 1994a). Other screenings have revealednumerous NGR-containing peptides (see Table 1). Despite the weakintegrin binding ability of NGR peptides, an integrin receptor may notbe the target molecule recognized by the NGR tumor homing peptidesexemplified herein (Example VII). As used herein, the term “integrin”means a heterodimeric cell surface adhesion receptor.

The peptides expressed by the phage that homed to the breast tumorincluded the peptides CGRECPRLCQSSC (SEQ ID NO: 2) and CNGRCVSGCAGRC(SEQ ID NO: 3; see Table 1; Example II). Similarly, tumor homingpeptides, including the peptides CDCRGDCFC (SEQ ID NO: 1) and CGSLVRC(SEQ ID NO: 5), were identified from two other phage librariesadministered to breast tumor bearing mice (Table 1). Some of thesemotifs, as well as novel one, also were isolated in the screen withmouse melanoma and human Kaposi's sarcoma (see Tables 2 and 3). Theseresults demonstrated that tumor homing molecules can be identified usingin vivo panning.

Three main tumor homing motifs emerged. As discussed above, one motifcontained the sequence RGD (Ruoslahti, supra, 1996) embedded in thepeptide structure, CDCRGDCFC (SEQ ID NO: 1), which is known to bindselectively to α_(v)-integrins (Koivunen et al., supra, 1995; WO95/14714). Since the α_(v)β₃ and α_(v)β₅ integrins are markers ofangiogenic vessels (Brooks et al., supra, 1994; Friedlander et al.,Science 270:1500 (1995)), a phage expressing the peptide CDCRGDCFC (SEQID NO: 1) was examined for tumor targeting and, as disclosed herein,homed to tumors in a highly selective manner (see Example III).Furthermore, homing by the CDCRGDCFC (SEQ ID NO: 1) phage was inhibitedby coadministration of the free CDCRGDCFC (SEQ ID NO: 1) peptide.

Another breast tumor homing peptide had the sequence CNGRCVSGCAGRC (SEQID NO: 3), which contains the NGR motif previously shown to have weakintegrin binding activity (Koivunen et al., J. Biol. Chem.268:20205-20210 (1993); Koivunen et al., supra, 1994a; WO 95/14714).Since an NGR containing peptide was identified, two additional peptides,the linear peptide, NGRAHA (SEQ ID NO: 6), and the cyclic peptide,CVLNGRMEC (SEQ ID NO: 7), each of which contains the NGR motif, wereexamined for tumor homing. Like the phage expressing CNGRCVSGCAGRC (SEQID NO: 3), phage expressing NGRAHA (SEQ ID NO: 6) or CVLNGRMEC (SEQ IDNO: 7) homed to the tumors. Furthermore, tumor homing was not dependenton the tumor type or on species, as the phage accumulated selectively inhuman breast carcinoma, as well as in the tumors of mice bearing a mousemelanoma and mice bearing a human Kaposi's sarcoma xenograft.

The various peptides, including RGD- and NGR-containing peptides,generally were bound to the tumor blood vessels. The minimal cyclic NGRpeptide, CNGRC (SEQ ID NO: 8), was synthesized based on theCNGRCVSGCAGRC (SEQ ID NO: 3) sequence. When the CNGRC (SEQ ID NO: 8)peptide was co-injected with phage expressing either CNGRCVSGCAGRC (SEQID NO: 3), NGRAHA (SEQ ID NO: 6) or CVLNGRMEC (SEQ ID NO: 7),accumulation of the phage in the breast carcinoma xenografts wasinhibited. However, the CNGRC (SEQ ID NO: 8) peptide did not inhibit thehoming of phage expressing the CDCRGDCFC (SEQ ID NO: 1) peptide, evenwhen administered in amounts up to ten times higher than those thatinhibited the homing of the NGR phage. In comparison, the CDCRGDCFC (SEQID NO: 1) peptide partially inhibited the homing of the NGR phage,although the amount needed was 5 to 10 fold higher than that of theCNGRC peptide (SEQ ID NO: 8). These results indicate that NGR peptidesand RGD peptides bind to different receptor sites in tumor vasculature.

A third motif, GSL (glycine-serine-leucine), also was identifiedfollowing in vivo panning in mice bearing breast carcinoma, malignantmelanoma or Kaposi's sarcoma. Homing of phage expressing the GSLpeptide, CGSLVRC (SEQ ID NO: 5), was inhibited by coadministration ofthe free CGSLVRC (SEQ ID NO: 5) peptide. Like the RGD and NGR peptides,phage expressing GSL peptides also bound to blood vessels of tumors. Inview of the identification of the conserved RGD, NGR and GSL motifspresent in tumor homing peptides, as disclosed herein, it will berecognized that peptides containing such motifs can be useful as tumorhoming peptides and, in particular, for forming conjugates that cantarget a moiety such as a cancer chemotherapeutic agent or a diagnosticagent to a tumor.

Various peptide libraries containing up to 13 amino acids wereconstructed and the NGR peptide, CNGRCVSGCAGRC (SEQ ID NO: 3), wasobtained as a result of in vivo panning against a breast tumor. This NGRpeptide, which was obtained by screening a random peptide library, was atumor homing peptide (see Example VII). In addition, when a peptidelibrary was constructed based on the formula CXXXNGRXX (SEQ ID NO: 13)or CXXCNGRCX (SEQ ID NO: 14), each of which is biased toward NGRsequences, and used for in vivo panning against a breast tumor, numerousNGR peptides were obtained (see Table 1).

These results indicate that a tumor homing peptide of the invention cancomprise the amino acid sequence RGD or NGR or GSL. Such tumor peptidescan be as small as five amino acids, such as CNGRC (SEQ ID NO: 8). Suchtumor homing peptides also can be not only at least 13 amino acids inlength, which is the largest peptide exemplified herein, but can be upto 20 amino acids, or 30 amino acids, or 50 to 100 amino acids inlength, as desired. A tumor homing peptide of the invention convenientlyis produced by chemical synthesis.

Immunohistochemical analysis was performed by comparing tissue stainingfor phage allowed to circulate for about four minutes, followed byperfusion through the heart of the mice, or with tissues analyzed 24hours after phage injection (see FIG. 1). At 24 hours followingadministration, essentially no phage remain in the circulation and,therefore, perfusion is not required (Pasqualini et al., supra, 1997).Strong phage staining was observed in tumor vasculature, but not innormal endothelium, in samples examined four minutes afteradministration of the CNGRCVSGCAGRC (SEQ ID NO: 3) phage (Example IV;compare FIGS. 1E, 1G, 1H and 1J). In comparison, staining of the tumorwas strong at 24 hours and appeared to have spread outside the bloodvessels into the tumor parenchyma (compare FIGS. 1A to 1D and 1F (tumor)with FIGS. 1I and 1K to 1V (nontumor)). The NGRAHA (SEQ ID NO: 6) andCVLNGRMEC (SEQ ID NO: 7) phage showed similar staining patterns (ExampleIV). In contrast, the control organs and tissues showed little or noimmunostaining, confirming the specificity of the NGR motifs for tumorvessels. Spleen and liver, however, captured phage, as expected, sinceuptake by the reticuloendothelial system is a general property of phageparticles, independent of the presence of peptide expression by thephage (Pasqualini et al., supra, 1997).

Immunostaining also was observed following administration of phageexpressing the GSL motif containing peptide, CLSGSLSC (SEQ ID NO: 4),and, like that of the NGR peptides, was localized to the blood vessels,in this case, within a melanoma tumor (see below; see, also, Examples IVand V). Similarly, immunostaining following administration of phageexpressing the RGD motif containing peptide, CDCRGDCFC (SEQ ID NO: 1),to breast tumor bearing mice was localized to the blood vessels in thetumor, but was not observed in brain, kidney or various other nontumortissues (see Examples III and IV; see, also, Pasqualini et al., supra,1997). These results demonstrate that the various tumor homing peptidesgenerally home to tumor vasculature.

The general applicability of the in vivo panning method for identifyingmolecules that home to a tumor was examined by injecting mice bearing asyngeneic melanoma with phage expressing a diverse population ofpeptides (Example V). The B16 mouse melanoma model was selected forthese studies because the tumors that form are highly vascularized andbecause the biology of this tumor line has been thoroughly characterized(see Miner et al., Cancer Res. 42:4631-4638 (1982)). Furthermore,because the B16 melanoma cells are of mouse origin, species differencesbetween the host and the tumor cell donor will not affect, for example,the distribution of phage into the tumor as compared to into normalorgans. As disclosed herein, in vivo panning against B16 melanoma cellsrevealed tumor homing peptides, including, for example, the GSL moietycontaining peptide CLSGSLSC (SEQ ID NO: 4; see, also, Table 2) andimmunohistochemical staining of the tumor and other organs using ananti-phage antibody demonstrated that the CLSGSLSC (SEQ ID NO: 4)expressing phage resulted in immunostaining in the melanoma, butessentially no staining in skin, kidney or other control organs (ExampleV). The staining pattern generally followed the blood vessels within themelanoma, but was not strictly confined to the blood vessels.

Although in vivo panning was performed in mice, at least the peptidescomprising an NGR, RGD or GSL motif also likely can target humanvasculature. The NGR phage binds to blood vessels in the transplantedhuman breast tumor, but not to blood vessels in normal tissues,indicating that this motif can be particularly useful for tumortargeting in patients. The CDCRGDCFC (SEQ ID NO: 1) peptide binds tohuman α_(v)-integrins (Koivunen et al., supra, 1995), which areselectively expressed in tumor blood vessels of human patients (Max etal., Int. J. Cancer 71:320 (1997); Max et al., Int. J. Cancer 72:706(1997)). Use of a moiety/CDCRGDCFC (SEQ ID NO: 1) conjugate to targetthe moiety to a tumor also provides the additional advantage that themoiety will be targeted to tumor cells, themselves, because breastcarcinoma cells, for example, can express the α_(v)β₃, integrin(Pasqualini et al., supra, 1997). In fact, many human tumors expressthis integrin, which may be involved in the progression of certaintumors such as malignant melanomas (Albelda et al., Cancer Res.50:6757-6764 (1990); Danen et al., Int. J. Cancer 61:491-496 (1995);Felding-Habermann et al., J. Clin Invest. 89:2018-2022 (1992); Sanderset al., Cold Spring Harb. Symp. Quant. Biol. 58:233-240 (1992); Mitjanset al., J. Cell. Sci. 108:3067-3078 (1995)). Unlike the CDCRGDCFC (SEQID NO: 1) peptide, the NGR peptides do not appear to bind to MDA-MD-435breast carcinoma cells. However, NGR peptides were able to deliver atherapeutically effective amount of doxorubicin to breast tumors,(Example VII), indicating that, even where a tumor homing molecule homesonly to tumor vasculature, i.e., not directly to the tumor cells, suchvasculature targeting in sufficient to confer the effect of the moietylinked to the molecule.

Since the α_(v)β₃ integrin is expressed by endothelial cells inangiogenic vasculature, experiments were performed to determine whethertumor vasculature that is undergoing angiogenesis can be targeted invivo using methods as disclosed herein. Phage expressing the peptide,CDCRGDCFC (SEQ ID NO: 1; see, Koivunen et al., supra, 1995), which isknown to bind to the α_(v)β₃ integrin, were injected into mice bearingtumors formed from human breast carcinoma cells, mouse melanoma cells orhuman Kaposi's sarcoma cells (see Example IV). The CDCRGDCFC (SEQ IDNO: 1) phage selectively homed to each of the tumors, whereas suchhoming did not occur with control phage. For example, in mice bearingtumors formed by implantation of human breast carcinoma cells, a twenty-to eighty-fold greater number of the CDCRGDCFC (SEQ ID NO: 1) phage, ascompared to unselected control phage, accumulated in the tumor.

Tissue staining for the phage showed accumulation of the CDCRGDCFC (SEQID NO: 1) phage in the blood vessels within the tumor, whereas nostaining was observed in brain, kidney or other control organs.Specificity of tumor homing by the CDCRGDCFC (SEQ ID NO: 1) phage wasdemonstrated by competition experiments, in which coinjection of thefree CDCRGDCFC (SEQ ID NO: 1) peptide greatly reduced tumor homing ofthe RGD phage, whereas coinjection of a non-RGD-containing controlpeptide had no effect on homing of the RGD phage (see Example III).These results demonstrate that the α_(v)β₃ target molecule is expressedon the luminal surface of endothelial cells in a tumor and that apeptide that binds to an α_(v)-containing integrin can bind selectivelyto this integrin and, therefore, to vasculature undergoing angiogenesis.

The results of these studies indicate that tumor homing molecules can beidentified by in vivo panning and that, in some cases, a tumor homingmolecule can home to vascular tissue in the tumor as well as to tumorparenchyma, probably due to the fenestrated nature of the blood vesselspermitting ready exit of the phage from the circulatory system. Due tothe ability of such tumor homing molecules to home to tumors, themolecules are useful for targeting a linked moiety to tumors. Thus, theinvention provides conjugates comprising a tumor homing molecule linkedto a moiety, such conjugates being useful for targeting the moiety totumor cells.

The ability of a molecule that homes to a particular tumor toselectively home to another tumor of the same or a similar histologictype can be determined using, for example, human tumors grown in nudemice or mouse tumors grown in syngeneic mice for these experiments. Forexample, various human breast cancer cell lines, including MDA-MB-435breast carcinoma (Price et al., Cancer Res. 50:717-721 (1990)),SKBR-1-II and SK-BR-3 (Fogh et al., J. Natl. Cancer Inst. 59:221-226(1975)), and mouse mammary tumor lines, including EMT6 (Rosen et al.,Int. J. Cancer 57:706-714 (1994)) and C3-L5 (Lala and Parhar, Int. J.Cancer 54:677-684 (1993)), are readily available and commonly used asmodels for human breast cancer. Using such breast tumor models, forexample, information relating to the specificity of an identified breasttumor homing molecule for diverse breast tumors can be obtained andmolecules that home to a broad range of different breast tumors orprovide the most favorable specificity profiles can be identified. Inaddition, such analyses can yield new information, for example, abouttumor stroma, since stromal cell gene expression, like that ofendothelial cells, can be modified by the tumor in ways that cannot bereproduced in vitro.

Selective homing of a molecule such as a peptide or protein to a tumorcan be due to specific recognition by the peptide of a particular celltarget molecule such as a cell surface receptor present on a cell in thetumor. Selectivity of homing is dependent on the particular targetmolecule being expressed on only one or a few different cell types, suchthat the molecule homes primarily to the tumor. As discussed above, theidentified tumor homing peptides, at least in part, can recognizeendothelial cell surface markers in the blood vessels present in thetumors. However, most cell types, particularly cell types that areunique to an organ or tissue, can express unique target molecules. Thus,in vivo panning can be used to identify molecules that selectively hometo a particular type of tumor cell such as a breast cancer cell andspecific homing can be demonstrated by performing the appropriatecompetition experiments.

A tumor homing molecule of the invention can be used to target a moietyto a tumor by linking the moiety to the molecule to produce a tumorhoming molecule/moiety conjugate and administering the conjugate to asubject having a tumor. As used herein, the term “moiety” is usedbroadly to mean a physical, chemical, or biological material that islinked to a tumor homing molecule for the purpose of being targeted invivo to a tumor or to angiogenic vasculature expressing a targetmolecule recognized by the tumor homing molecule. In particular, amoiety is a biologically useful moiety such as therapeutic moiety, adiagnostic moiety or a drug delivery vehicle. Thus, a moiety can be atherapeutic agent, for example, a cancer chemotherapeutic agent such asdoxorubicin, which, when linked to a tumor homing molecule, provides aconjugate useful for treating a cancer in a subject. In addition, amoiety can be a drug delivery vehicle such as a chambered microdevice, acell, a liposome or a virus, which can contain an agent such as a drugor a nucleic acid.

A moiety also can be a molecule such as a polypeptide or nucleic acid,to which a tumor homing molecule is grafted for the purpose of directingthe polypeptide or nucleic acid to a selected tumor (Smith et al., J.Biol. Chem. 269:32788-32795 (1994); Goldman et al., Cancer Res.15:1447-1451 (1997), each of which is incorporated herein by reference).For example, a peptide tumor homing molecule can be expressed as afusion protein with a desired polypeptide such that the peptide targetsthe grafted polypeptide to a selected tumor. Such a desired polypeptide,which is grafted to the tumor homing peptide, can be a polypeptideinvolved in initiating a cell death pathway, for example, caspase 8,thus providing a means to direct caspase 8 to a tumor, where it caninduce apoptosis of the tumor cells or of the vasculature supplying thetumor. A tumor homing peptide also can be grafted to a polypeptideexpressed by a virus, for example, the adenovirus penton base coatprotein, thus providing a means to target a virus to a tumor (Wickham etal., Gene Ther. 2:750-756 (1995); Weitzman et al., In: “Gene Therapy andVector Systems” 2:17-25 (1997), each of which is incorporated herein byreference; see, also, Example III). Such a grafted virus can contain anexogenous gene useful in a method of gene therapy. Accordingly, theinvention provides compositions of matter comprising a tumor homingmolecule/moiety conjugate.

A moiety can be a detectable label such a radiolabel or can be acytotoxic agent, including a toxin such as ricin or a drug such as achemotherapeutic agent or can be a physical, chemical or biologicalmaterial such as a liposome, microcapsule, micropump or other chamberedmicrodevice, which can be used, for example, as a drug delivery system.Generally, such microdevices, should be nontoxic and, if desired,biodegradable. Various moieties, including microcapsules, which cancontain an agent, and methods for linking a moiety, including achambered microdevice, to a molecule of the invention are well known inthe art and commercially available (see, for example, “Remington'sPharmaceutical Sciences” 18th ed. (Mack Publishing Co. 1990), chapters89-91; Harlow and Lane, Antibodies: A laboratory manual (Cold SpringHarbor Laboratory Press 1988), each of which is incorporated herein byreference; see, also, Hermanson, supra, 1996).

As disclosed herein, a moiety can be, for example, a cancerchemotherapeutic agent linked to a tumor homing molecule to produce atumor homing molecule/moiety conjugate. Cytotoxic chemotherapy is thebasis of the systemic treatment of disseminated malignant tumors.However, a major limitation of the currently used chemotherapeuticagents is that these drugs have the narrowest therapeutic index in allof medicine. As such, the dose of cancer chemotherapeutic agentsgenerally is limited by undesirable toxicity to the patient beingtreated. Thus, the ability of tumor homing peptides of the invention totarget drugs into tumors was examined. As disclosed herein, the linkingof a cancer chemotherapeutic agent, doxorubicin, to a tumor homingmolecule reduced the systemic toxicity of the doxorubicin and enhancedanti-tumor activity of the agent (see Example VII).

A conjugate of the invention is exemplified herein by doxorubicin linkedto various tumor homing peptides (see Examples VI and VII). In view ofthe exemplified method of linking doxorubicin to various tumor homingpeptides and the disclosed efficacy of such conjugates of the invention,the skilled artisan will recognize that various other chemotherapeuticagents also can be linked to a tumor homing molecule to make a conjugateof the invention. Cancer chemotherapeutic agents have been linked toantibodies, for example, for the purpose of targeting the agents tocells such as tumor cells that express the antigen recognized by theantibodies. In addition, in such antibody/drug conjugates, the agent canmaintain its therapeutic function and the antibody can maintain itsantigen binding specificity. For example, the anthracyclin, doxorubicin,has been linked to antibodies and the antibody/doxorubicin conjugateshave been therapeutically effective in treating tumors (Sivam et al.,Cancer Res. 55:2352-2356 (1995); Lau et al., Bioorg. Med. Chem.3:1299-1304 (1995); Shih et al., Cancer Immunol. Immunother. 38:92-98(1994)). Similarly, other anthracyclins, including idarubicin anddaunorubicin, have been chemically conjugated to antibodies, which havedelivered effective doses of the agents to tumors (Rowland et al.,Cancer Immunol. Immunother. 37:195-202 (1993); Aboud-Pirak et al.,Biochem. Pharmacol. 38:641-648 (1989)).

In addition to the anthracyclins, alkylating agents such as melphalaiiand chlorambucil have been linked to antibodies to producetherapeutically effective conjugates (Rowland et al., supra, 1994; Smythet al., Imununol. Cell Biol 65:315-321 (1987)), as have vinca alkaloidssuch as vindesine and vinblastine (Aboud-Pirak et al., supra, 1989;Starling et al., Bioconj. Chem. 3:315-322 (1992)). Similarly, conjugatesof antibodies and antimetabolites such as 5-fluorouracil,5-fluorouridine and derivatives thereof have been effective in treatingtumors (Krauer et al., Cancer Res. 52:132-137 (1992); Henn et al., J.Med. Chem. 36:1570-1579 (1993)). Other chemotherapeutic agents,including cis-platinum (Schechter et al., Int. J. Cancer 48:167-172(1991)), methotrexate (Shawler et al., J. Biol. Resp. Mod. 7:608-618(1988); Fitzpatrick and Garnett, Anticancer Drug Des. 10:11-24 (1995))and mitomycin-C (Dillman et al., Mol. Biother. 1:250-255 (1989)) alsoare therapeutically effective when administered as conjugates withvarious different antibodies.

The results obtained using antibody/drug conjugates demonstrate that achemotherapeutic agent can be linked to an antibody to produce aconjugate that maintains the antigen binding specificity of the antibodyand the therapeutic function of the agent. As disclosed herein, aconjugate comprising doxorubicin and a tumor homing peptide maintainsthe tumor homing specificity of the tumor homing peptide as well as thetherapeutic efficacy of the chemotherapeutic agent (see Example VII).Such results are remarkable, since, in the doxorubicin/CNGRC. (SEQ IDNO: 8) conjugate, for example, the doxorubicin component has only aslightly lower molecular weight than the peptide and comprises about 46%of the molecular weight of the conjugate.

Since the moiety component of a tumor homing molecule/moiety conjugatecan comprise a substantial portion of the conjugate without adverselyaffecting the ability of the tumor homing molecule to home to a tumor,additional components can be included as part of the conjugate, ifdesired. For example, in some cases, it can be desirable to utilize anoligopeptide spacer between a tumor homing peptide and the moiety(Fitzpatrick and Garnett, Anticancer Drug Des. 10:1-9 (1995)). In thisway, panels of moiety/spacer complexes can be constructed, in which acommon spacer is linked to various different moieties. Such panels ofmoiety/spacer conjugates can facilitate linkage of the moiety to a tumorhoming molecule such as a tumor homing peptide of choice.

Doxorubicin is one of the most commonly used cancer chemotherapeuticagents and, particularly, is used for treating breast cancer (Stewartand Ratain, In: “Cancer: Principles and practice of oncology” 5th ed.chap. 19 (eds. DeVita, Jr., et al.; J. P. Lippincott 1997); Harris etal., In “Cancer: Principles and practice of oncology,” supra, 1997). Inaddition, doxorubicin has anti-angiogenic activity (Folkman, supra,1997; Steiner, In “Angiogenesis: Key principles-Science, technology andmedicine,” pp. 449-454 (eds. Steiner et al.; Birkhauser Verlag, 1992)),which can contribute to its effectiveness in treating cancer. Thus,treatment of human breast cancer xenografts in mice using doxorubicinwas selected as a model for exemplifying the present invention.

CDCRGDCFC (SEQ ID NO: 1) and CNGRC (SEQ ID NO: 8) were coupled todoxorubicin (Example VI) and the peptide/doxorubicin conjugates wereused to treat mice bearing tumors derived from human MDA-MB-435 breastcarcinoma cells (Example VII). Mice were treated with 5 μg/week ofdoxorubicin equivalent (i.e., either free doxorubicin or the doxorubicincomponent of the peptide/doxorubicin conjugate), as compared to the morecommonly used 50-200 μg/mouse used in tumor bearing mice (Berger et al.,In “The Nude Mouse in Oncology Research” (CRC Press 1991)). The lowerdose was selected because it was expected that the conjugate would bemore effective than the free drug.

MDA-MB-435 tumor-bearing mice treated with the doxorubicin/CDCRGDCFC(SEQ ID NO: 1) conjugate had significantly smaller tumors, less spreadto regional lymph nodes, and fewer pulmonary metastasis than micetreated with free doxorubicin (see Example VII). All of the mice treatedwith the doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate survived beyondthe time when all of the mice treated with free doxorubicin had diedfrom widespread disease. In a dose-escalation experiment, the tumorbearing mice were treated with the doxorubicin/CDCRGDCFC (SEQ ID NO: 1)conjugate at 30 μg/mouse every three weeks for three cycles, then wereobserved, without further treatment, for an extended period of time. Theconjugate treated mice all remained alive more than 6 months after thecontrol, doxorubicin treated mice had died (Example VII). These resultsindicate that primary tumor growth and metastasis significantly wereinhibited in mice treated with the conjugate and that cures may haveoccurred.

Many of the mice that received doxorubicin/CDCRGDCFC (SEQ ID NO: 1)conjugate presented marked skin ulceration and tumor necrosis; no suchsigns were observed in mice treated with free doxorubicin or withdoxorubicin conjugated to an unrelated peptide (Example VII).Histopathological analysis disclosed a pronounced destruction of thevasculature in the tumors treated with conjugate as compared to micetreated with free doxorubicin. Furthermore, when tumors were removedfrom the mice and the tumor cells plated in culture, viability of cellsfrom the tumors of mice receiving the doxorubicin/CDCRGDCFC (SEQ IDNO: 1) conjugate was about 3 fold less than cells from tumors of micetreated with the free doxorubicin (see Example VII). These resultsdemonstrate that administration to a tumor bearing mouse of a conjugatecomprising a chemotherapeutic agent linked to a tumor homing molecule ismore efficacious than administration of the agent, alone, in treating atumor.

Toxicity was determined by administration of 200 μg/doxorubicinequivalent in mice with very large, size matched breast tumors. All ofthe mice treated with the doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugatesurvived more than a week, while all of the mice treated with freedoxorubicin died within 48 hours of the administration of the drug(Example VII). These results indicate that accumulation of the tumorhoming peptide/doxorubicin conjugate in the large tumors can reducesystemic toxicity of the agent.

Similar toxicity and treatment efficacy results were obtained whenbreast tumor bearing mice were treated using a doxorubicin/CNGRC (SEQ IDNO: 8) conjugate. Tumors in the mice treated with the CNGRC (SEQ ID NO:8) conjugate were significantly smaller than in the control groups; theconjugate suppressed tumor growth-almost completely. A strong effect onsurvival also occurred. Free doxorubicin or doxorubicin conjugated to anunrelated peptide, at the dose used, had little if any effect on tumorgrowth relative to vehicle alone.

Cytotoxic activity of free doxorubicin and the doxorubicin/peptideconjugates was compared in vitro using MDA-MB-435 cells. When cells wereexposed to free doxorubicin, doxorubicin/CDCRGDCFC (SEQ ID NO: 1) ordoxorubicin conjugated to an unrelated peptide for 30 minutes, celldeath occurred only in the cultures treated with thedoxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate. In comparison, cellswere killed by all of the treatments after 24 hours of exposure. Theseresults indicate that enhanced cellular uptake of thedoxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate occurs.

As disclosed herein, tumor homing molecules of the invention can bind tothe endothelial lining of small blood vessels of tumors. The vasculaturewithin tumors is distinct, presumably due to the continualneovascularization, resulting in the formation of new blood vesselsrequired for tumor growth. The distinct properties of the angiogenicneovasculature within tumors are reflected in the presence of specificmarkers in endothelial cells and pericytes (Folkman, Nature Biotechnol.15:510 (1997); Risau, FASEB J. 9:926-933 (1995); Brooks et al., supra,1994); these markers likely are being targeted by the tumor homingmolecules of the invention.

The ability of a tumor homing molecule to target the blood vessels in atumor provides substantial advantages over methods of systemic treatmentor methods that directly target the tumor cells. For example, tumorcells depend on a vascular supply for survival and the endotheliallining of blood vessels is readily accessible to a circulating probe.Conversely, in order to reach solid tumor cells, a chemotherapeuticagent must overcome potentially long diffusion distances, closely packedtumor cells, and a dense fibrous stroma with a high interstitialpressure that impedes extravasation (Burrows and Thorpe, Pharmacol.Ther. 64:155-174 (1994)).

In addition, where the tumor vasculature is targeted, the killing of alltarget cells may not be required, since partial denudation of theendothelium can lead to the formation of an occlusive thrombus haltingthe blood flow through the entirety of the affected tumor vessel(Burrows and Thorpe, supra, 1994). Furthermore, unlike direct tumortargeting, there is an intrinsic amplification mechanism in tumorvasculature targeting. A single capillary loop can supply nutrients toup to 100 tumor cells, each of which is critically dependent on theblood supply (Denekamp, Cancer Metast. Rev. 9:267-282 (1990); Folkman,supra, 1997).

Endothelial cells in a tumor also are unlikely to lose a cell surfacetarget receptor or develop a drug resistance phenotype, as can developthrough mutation and clonal evolution of tumor cells, becauseendothelial cells are genetically stable despite their highproliferation rates (Burrows and Thorpe, supra, 1994; Folkman, supra,1995; Folkman, supra, 1997). In this regard, it has been long recognizedby medical oncologists that, while tumors treated with chemotherapeuticagents commonly develop drug resistance, normal tissues such as bonemarrow do not develop such resistance. Thus, toxicity to normal tissuessuch as chemotherapy induced myelosuppression continues to occur duringa treatment, even after tumor cells have become drug resistant. Sincethe endothelial cells in blood vessels supplying a tumor are nontumorcells, it is expected that they will not develop resistance tochemotherapeutic agents, in a manner analogous to bone marrow cells. Infact, drug resistance has not been observed during long termanti-angiogenic therapy in either experimental animals or in clinicaltrials (Folkman, supra, 1997).

Linking of a moiety larger than an agent such as a drug or other organicor biologic molecule to a tumor homing molecule for the purpose ofdirecting homing of the moiety to the selected tumor is exemplified byexpressing an RGD-containing peptide on a phage, wherein the peptidedirected homing of the phage to breast tumor vasculature (Example IV).These results indicate that a tumor homing molecule of the invention canbe linked to other moieties including, for example, a chamberedmicrodevice or a liposome or a cell such as a white blood cell (WBC),which can be a cytotoxic T cell or a killer cell, wherein uponadministration of the tumor homing molecule/WBC conjugate, the moleculedirects homing of the WBC to the tumor, where the WBC can exert itseffector function.

The linking of a moiety to a tumor homing molecule can result in themolecule directing homing of the linked moiety to a tumor. For example,the linking of a brain homing peptide to a RBC directed homing of theRBC to brain (see U.S. Pat. No. 5,622,699; Pasqualini and Ruoslahti,supra, 1996). This result indicates that a tumor homing molecule of theinvention also can be linked to cell type or to a physical, chemical orbiological delivery system such as a liposome or other encapsulatingdevice, which can contain an agent such as drug, in order to direct thecell type or the delivery system to a selected tumor. For example, atumor homing molecule identified by in vivo panning can be linked to awhite blood cell (WBC) such as a cytotoxic T cell or a killer cell,wherein upon administration of the tumor homing molecule/WBC conjugate,the molecule directs homing of the WBC to the tumor, where the WBC canexert its effector function. Similarly, a tumor homing molecule can belinked to a liposome or to a chambered microdevice comprising, forexample, a permeable or semipermeable membrane, wherein an agent such asa drug to be delivered to a selected tumor is contained within theliposome or microdevice. Such compositions also can be useful, forexample, for delivering a nucleic acid molecule to a tumor cells,thereby providing a means for performing in vivo targeted gene therapy.

In one embodiment a tumor homing molecule is linked to a moiety that isdetectable external to the subject, thereby providing a compositionuseful to perform an in vivo diagnostic imaging study. For example, invivo imaging using a detectably labeled tumor homing peptide canidentify the presence of a tumor in a subject. For such studies, amoiety such as a gamma ray emitting radionuclide, for example,indium-111 or technitium-99, can be linked to the tumor homing moleculeand, following administration to a subject, can be detected using asolid scintillation detector. Similarly, a positron emittingradionuclide such as carbon-11 or a paramagnetic spin label such ascarbon-13 can be linked to the molecule and, following administration toa subject, the localization of the moiety/molecule can be detected usingpositron emission transaxial tomography or magnetic resonance imaging,respectively. Such methods can identify a primary tumor as well as ametastatic lesion, which may not be detectable using other methods.Having identified the presence of a cancer in a subject, in anotherembodiment of the invention, the tumor homing molecule is linked to acytotoxic agent such as ricin or a cancer chemotherapeutic agent such asdoxorubicin in order to direct the moiety to the tumor or can be linkedto a chambered microdevice, which can contain a chemotherapeutic drug orother cytotoxic agent. Use of such a composition provides a means toselectively killing of the tumor, while substantially sparing normaltissues in a cancer patient and, therefore, the conjugates of theinvention provide useful medicaments for diagnosing or treating a cancerpatient.

The skilled artisan would recognize that various tumor homing moleculescan selectively home only to a tumor or can selectively home to a tumorand to a family of selected organs, including, in some cases, the normaltissue counterpart to the tumor. Thus, the artisan would select a tumorhoming peptide for administration to a subject based on the procedurebeing performed. For example, a tumor homing molecule that homes only toa tumor can be useful for directing a therapy to the tumor. Incomparison, a tumor homing molecule that selectively homes not only tothe tumor, but also to one or more normal organs or tissues, can be usedin an imaging method, whereby homing to an organ or tissue other thanthe tumor provides an internal imaging control. Such an internal controlcan be useful, for example, for detecting a change in the size of atumor in response to a treatment, since the normal organ is not expectedto change in size and, therefore, can be compared with the tumor size.

Tumor homing peptides, which are identified by in vivo panning, can besynthesized in required quantities using routine methods of solid statepeptide synthesis or can be purchased from commercial sources (forexample, Anaspec; San Jose Calif.) and a desired moiety can be linked tothe molecule. Several methods useful for linking a moiety to a moleculeare known in the art, depending on the particular chemicalcharacteristics of the molecule. For example, methods of linking haptensto carrier proteins as used routinely in the field of applied immunology(see, for example, Harlow and Lane, supra, 1988; Hermanson, supra,1996).

It is recognized that, in some cases, a drug can lose cytotoxic efficacyupon conjugation or derivatization depending, for example, on theconjugation procedure or the chemical group utilized (Hurwitz et al.,Cancer Res. 35:1175-1181 (1975); Trail et al., Science 261; 212-215(1993); Nagy et al., Proc. Natl. Acad. Sci. USA 93:7269-7273 (1996)).Moreover, it is recognized that a phage that yields a tumor homingpeptide of the invention displays as many as five of the peptides. Thus,there is a possibility that the affinity of an individual peptide is toolow for effective tumor homing and that multivalent, rather thanunivalent, peptide conjugates must be used. However, as disclosedherein, doxorubicin maintained cytotoxic activity when used as aconjugate with tumor homing peptides (see Example VII), thus allayingthe potential concerns discussed above.

A moiety such as a therapeutic or diagnostic agent can be conjugated toa tumor homing peptide using, for example, carbodiimide conjugation(Bauminger and Wilchek, Meth Enzymol. 70:151-159 (1980), which isincorporated herein by reference). Carbodiimides comprise a group ofcompounds that have the general formula R—N═C═N—R′, where R and R′ canbe aliphatic or aromatic, and are used for synthesis of peptide bonds.The preparative procedure is simple, relatively fast, and is carried outunder mild conditions. Carbodiimide compounds attack carboxylic groupsto change them into reactive sites for free amino groups. Carbodiimideconjugation has been used to conjugate a variety of compounds tocarriers for the production of antibodies.

The water soluble carbodiimide,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is particularlyuseful for conjugating a moiety to a tumor homing peptide and was usedto conjugate doxorubicin to tumor homing peptides (Example VI). Theconjugation of doxorubicin and a tumor homing peptide requires thepresence of an amino group, which is provided by doxorubicin, and acarboxyl group, which is provided by the peptide. EDC coupling ofdoxorubicin to the CNGRC (SEQ ID NO: 8) peptide was performed using a1:1 molar ratio of the peptide (carboxyl groups) to obtain adoxorubicin/CNGRC (SEQ ID NO: 8; see Example VI).

In addition to using carbodiimides for the direct formation of peptidebonds, EDC also can be used to prepare active esters such asN-hydroxysuccinimide (NHS) ester. The NHS ester, which binds only toamino groups, then can be used to induce the formation of an amide bondwith the single amino group of the doxorubicin. The use of EDC and NHSin combination is commonly used for conjugation in order to increaseyield of conjugate formation (Bauminger and Wilchek, supra, 1980).

Other methods for conjugating a moiety to a tumor homing molecule alsocan be used. For example, sodium periodate oxidation followed byreductive alkylation of appropriate reactants can be used, as canglutaraldehyde crosslinking. However, it is recognized that, regardlessof which method of producing a conjugate of the invention is selected, adetermination must be made that the tumor homing molecule maintains itstargeting ability and that the moiety maintains its relevant function.Methods as disclosed in Example VII or otherwise known in the art canconfirm the activity of the moiety/tumor homing molecule conjugate.

The yield of moiety/tumor homing molecule conjugate formed is determinedusing routine methods. For example, HPLC or capillary electrophoresis orother qualitative or quantitative method can be used (see, for example,Liu et al., J. Chromatogr. 735:357-366 (1996); Rose et al., J.Chromatogr, 425:419-412 (1988), each of which is incorporated herein byreference; see, also, Example V). In particular, the skilled artisanwill recognize that the choice of a method for determining yield of aconjugation reaction depends, in part, on the physical and chemicalcharacteristics of the specific moiety and tumor homing molecule.Following conjugation, the reaction products are desalted to remove anyfree peptide and free drug.

When administered to a subject, the tumor homing molecule/moietyconjugate is administered as a pharmaceutical composition containing,for example, the conjugate and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers are well known in the art andinclude, for example, aqueous solutions such as water or physiologicallybuffered saline or other solvents or vehicles such as glycols, glycerol,oils such as olive oil or injectable organic esters.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act, for example, to stabilize or to increasethe absorption of the conjugate. Such physiologically acceptablecompounds include, for example, carbohydrates, such as glucose, sucroseor dextrans, antioxidants, such as ascorbic acid or glutathione,chelating agents, low molecular weight proteins or other stabilizers orexcipients. One skilled in the art would know that the choice of apharmaceutically acceptable carrier, including a physiologicallyacceptable compound, depends, for example, on the route ofadministration of the composition. The pharmaceutical composition alsocan contain an agent such as a cancer therapeutic agent.

One skilled in the art would know that a pharmaceutical compositioncontaining a tumor homing molecule can be administered to a subject byvarious routes including, for example, orally or parenterally, such asintravenously. The composition can be administered by injection or byintubation. The pharmaceutical composition also can be a tumor homingmolecule linked to liposomes or other polymer matrices, which can haveincorporated therein, for example, a drug such as a chemotherapeuticagent (Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton,Fla. 1984), which is incorporated herein by reference). Liposomes, forexample, which consist of phospholipids or other lipids, are nontoxic,physiologically acceptable and metabolizable carriers that arerelatively simple to make and administer.

For the diagnostic or therapeutic methods disclosed herein, an effectiveamount of the tumor homing molecule/moiety conjugate must beadministered to the subject. As used herein, the term “effective amount”means the amount of the conjugate that produces the desired effect. Aneffective amount often will depend on the moiety linked to the tumorhoming molecule. Thus, a lesser amount of a radiolabeled molecule can berequired for imaging as compared to the amount of a drug/moleculeconjugate administered for therapeutic purposes. An effective amount ofa particular molecule/moiety for a specific purpose can be determinedusing methods-well known to those in the art.

The route of administration of a tumor homing molecule will depend, inpart, on the chemical structure of the molecule. Peptides, for example,are not particularly useful when administered orally because they can bedegraded in the digestive tract. However, methods for chemicallymodifying peptides to render them less susceptible to degradation byendogenous proteases or more absorbable through the alimentary tract arewell known (see, for example, Blondelle et al., supra, 1995; Ecker andCrooke, supra, 1995; Goodman and Ro, supra, 1995). Such modificationscan be performed on peptides identified by in vivo panning. In addition,methods for preparing libraries of peptidomimetics, which can containD-amino acids, other non-naturally occurring amino acids, or chemicallymodified amino acids; or can be organic molecules that mimic thestructure of peptide; or can be peptoids such as vinylogous peptoids,are known in the art and can be used to identify molecules that home toa tumor and are stable for oral administration.

Tumor homing molecules obtained using the methods disclosed herein alsocan be useful for identifying a target molecule such as a cell surfacereceptor or a ligand for a receptor, which is recognized by the tumorhoming peptide, or for substantially isolating the target molecule. Forexample, a tumor homing peptide can be linked to a solid support such asa chromatography matrix. The linked peptide then can be used foraffinity chromatography by passing an appropriately processed sample ofa tumor over the column in order to bind a particular target molecule.The target molecule, which forms a complex with the tumor homingmolecule, then can be eluted from the column and collected in asubstantially isolated form. The substantially isolated target moleculethen can be characterized using well known methods. A tumor homingpeptide also can be linked to a detectable moiety such as aradionuclide, a fluorescent molecule, an enzyme or biotin and can beused, for example, to screen a sample in order to detect the presence ofthe target molecule in a tumor or to follow the target molecule duringvarious isolation steps.

It follows that, upon identifying the presence of a target molecule in atumor sample, the skilled artisan readily can obtain the target moleculein a substantially isolated form. For example, the sample containing thetarget molecule can be passed over a column containing attached theretothe relevant tumor homing molecule, thereby providing a means to obtainthe target molecule in substantially isolated form. Thus, the inventionfurther provides a substantially isolated target molecule, whichspecifically binds a tumor homing molecule and which can be obtainedusing the methods disclosed herein.

The methods of the present invention were used to identify tumor homingpeptides, which can selectively home to various tumors. It should berecognized that cysteine residues were included in some peptides suchthat cyclization of the peptides could be effected. In fact, thepeptides containing at least two cysteine residues cyclizespontaneously. However, such cyclic peptides also can be active whenpresent in a linear form (see, for example, Koivunen et al., supra,1993) and, as disclosed herein, a linear peptide, NGRAHA (SEQ ID NO: 6),also was useful as tumor homing molecule (Example VII; see, also, Table1). Thus, in some cases one or more cysteine residues in the peptidesdisclosed herein or otherwise identified as tumor homing peptides can bedeleted without significantly affecting the tumor homing activity of thepeptide. Methods for determining the necessity of a cysteine residue orof amino acid residues N-terminal or C-terminal to a cysteine residuefor tumor homing activity of a peptide of the invention are routine andwell known in the art.

A tumor homing peptide is useful, for example, for targeting a desiredmoiety to the selected tumor as discussed above. In addition, a tumorhoming peptide can be used to identify the presence of a target moleculein a sample. As used herein, the term “sample” is used in its broadestsense to mean a cell, tissue, organ or portion thereof, including atumor, that is isolated from the body. A sample can be, for example, ahistologic section or a specimen obtained by biopsy or cells that areplaced in or adapted to tissue culture. If desired, a sample can beprocessed, for example, by homogenization, which can be an initial stepfor isolating the target molecule to which a tumor homing moleculebinds.

A tumor homing peptide such as a breast tumor homing peptide can be usedto identify the target molecule expressed in a breast tumor. Forexample, a breast tumor homing peptide can be attached to a matrix suchas a chromatography matrix to produce a peptide affinity matrix. Ahomogenized sample of a breast tumor can be applied to thepeptide-affinity matrix under conditions that allow specific binding ofthe target molecule to the tumor homing peptide (see, for example,Deutshcer, Meth, Enzymol., Guide to Protein Purification (AcademicPress, Inc., ed. M. P. Deutscher, 1990), Vol. 182, which is incorporatedherein by reference; see, for example, pages 357-379). Unbound andnonspecifically bound material can be removed and the specifically boundbreast tumor-derived target molecule can be isolated in substantiallypurified form. The presence or absence of the target molecule in normalbreast tissue also can be determined. Such an analysis can provideinsight into methods of treating the tumor.

As disclosed herein, a target molecule, which specifically binds a tumorhoming molecule, can be identified by contacting a sample of a tumorwith such a tumor homing molecule and identifying a target moleculebound by the tumor homing molecule. In parallel, the tumor homingmolecule is contacted with a sample of a nontumor tissue correspondingto the tumor. The presence of the target molecule in the tumor samplecan be identified by determining that the tumor homing molecule does notbind to a component of the corresponding nontumor tissue sample. Thus,the invention provides methods for identifying the presence of a targetmolecule, which is expressed in a tumor and specifically bound by atumor homing molecule.

Since numerous tumor homing peptides containing the NGR motif have beenidentified, for example, a tumor homing peptide comprising an NGRsequence can be used to isolate the NGR receptor. Thus, an NGR tumorhoming peptide can be linked to a solid matrix and an appropriatelyprocessed sample of a tumor, which specifically binds the NGR peptide,can be passed over the NGR peptide-matrix. The NGR receptor, which isthe target molecule for the NGR tumor homing peptide, then can beobtained in a substantially isolated form. When used in reference to atarget molecule, the term “substantially isolated” means that the targetmolecule comprises at least 30% of the total protein present, althoughthe target molecule can comprise at least 50% of the total protein, or80% of the total protein, or 90% or 95% of the total protein, or more. Amethod such as gel electrophoresis and silver staining can be used todetermine the relative amount of a target molecule in a sample,following a purification protocol, and, therefore, can be used toidentify a substantially isolated target molecule.

The skilled artisan will recognize that a substantially isolated targetmolecule can be used as an immunogen to obtain antibodies thatspecifically bind the target molecule. As used herein, the term“antibody” is used in its broadest sense to include polyclonal andmonoclonal antibodies, as well as antigen binding fragments of suchantibodies. With regard to an antibody of the invention, whichspecifically binds a target molecule targeted by a tumor homingmolecule, the term antigen means the target molecule polypeptide orpeptide portion thereof. An antibody or antigen binding fragment of anantibody that binds a target molecule is characterized by havingspecific binding activity for the target molecule or a peptide portionthereof of at least about 1×10⁵ M⁻¹, preferably at least about 1×10⁶ M⁻¹and more preferably at least about 1×10⁸ M⁻¹. Thus, Fab, F(ab′)₂, Fd andFv fragments of the antibody, which retain specific binding activity fora target molecule, which is expressed by angiogenic vasculature, areincluded within the definition of an antibody.

In addition, the term “antibody” as used herein includes naturallyoccurring antibodies as well as non-naturally occurring antibodies,including, for example, single chain antibodies, chimeric, bifunctionaland humanized antibodies, as well as antigen-binding fragments thereof.Such non-naturally occurring antibodies can be constructed using solidphase peptide synthesis, can be produced recombinantly or can beobtained, for example, by screening combinatorial libraries consistingof variable heavy chains and variable light chains as described by Huseet al., Science 246:1275-1281 (1989), which is incorporated herein byreference. These and other methods of making, for example, chimeric,humanized, CDR-grafted, single chain, and bifunctional antibodies arewell known to those skilled in the art (Winter and Harris, Immunol.Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Hilyardet al., Protein Engineering: A practical approach (IRL Press 1992);Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995);each of which is incorporated herein by reference; see, also, Harlow andLane, supra, 1988).

Antibodies that specifically bind a target molecule of the invention canbe raised using as an immunogen a substantially isolated targetmolecule, which can be obtained as disclosed herein, or a peptideportion of the target molecule, which can be obtained, for example, byenzymatic degradation of the target molecule and gel purification. Anon-immunogenic peptide portion of a target molecule can be; madeimmunogenic by coupling the hapten to a carrier molecule such as bovineserum albumin (BSA) or keyhole limpet hemocyanin (RLH). Various othercarrier molecules and methods for coupling a hapten to a carriermolecule are well known in the art and described, for example, by Harlowand Lane, supra, 1988).

Particularly useful antibodies of the invention are those that bind tothe tumor homing molecule binding site on the target molecule, suchantibodies being readily identifiable by detecting competitiveinhibition of binding of the antibody and the particular tumor homingmolecule that binds to the target molecule. Conversely, antibodies thatbind to an epitope of the target molecule that is not involved inbinding the tumor homing molecule also are valuable, since suchantibodies, which, themselves, can be “tumor homing molecules,” can bebind to target molecules having another tumor homing molecule boundthereto.

An antibody that specifically binds a target molecule, for example, theNGR receptor, is useful for determining the presence or level of thetarget molecule in a tissue sample, which can be a lysate or ahistological section. The identification of the presence or level of thetarget molecule in the sample can be made using well known immunoassayand immunohistochemical methods (Harlow and Lane, supra, 1988). Anantibody specific for a target molecule also can be used tosubstantially isolate the target molecule from a sample. In addition, anantibody of the invention can be used in a screening assay to identify,for example, peptidomimetics of a tumor homing molecule that bind to thetarget molecule or as-a tool for tumor targeting.

Upon obtaining a target molecule, which, due to the nature of a tumorhoming molecule, is expressed in angiogenic vasculature, for example,the angiogenic vasculature in a tumor, the naturally occurring ligandfor the target molecule, where it exists, can be identified. Methods foridentifying a ligand for such a target molecule, which is akin to an“orphan receptor,” are well known in the art and include, for example,screening biological samples to identify the ligand. A convenientscreening assay to identify a natural ligand for the target molecule canutilize the ability of a putative natural ligand to competitivelyinhibit the binding to the target molecule of a tumor homing moleculethat specifically binds the target molecule, for example, the tumorhoming peptide used to obtain the substantially isolated targetmolecule.

A screening assay comprising a competitive binding assay for the targetmolecule and, for example, the natural ligand for the target molecule ora tumor homing peptide that specifically binds the target molecule, alsoprovides a means to-identify peptidomimetics of a tumor homing molecule.As discussed above, such peptidomimetics can provide advantages overtumor homing peptides in that they can be small, relatively stable forstorage, conveniently produced in suitable quantities, and capable ofbeing administered orally. A peptidomimetic of a tumor homing peptidecan be identified by screening libraries of peptidomimetics in acompetitive binding assay as described above.

The disclosed in vivo panning method can be used to detect fourdifferent kinds of target molecules in tumors. First, because tumorvasculature undergoes active angiogenesis, target molecules that arecharacteristic of angiogenic vasculature, in general, or angiogenictumor vasculature, in particular, can be identified. Second, vasculartarget molecules that are characteristic of the tissue of origin of thetumor can be identified. Third, target molecules that are expressed inthe vasculature of a particular type of tumor can be identified. Fourth,tumor stroma or tumor cell target molecules can be identified due to thefenestrated nature of tumor vasculature, which allows the potentialtumor homing molecules to leave the circulation and contact the tumorparenchyma.

As further disclosed herein, some, but not all, tumor homing moleculesalso can home to angiogenic vasculature that is not contained within atumor. For example, tumor homing molecules containing either the RGDmotif or the GSL motif specifically homed to retinal neovasculature(Smith et al., Invest. Ophthamol. Vis. Sci. 35:101-111 (1994), which isincorporated herein by reference), whereas tumor homing peptidescontaining the NGR motif did not accumulate substantially to thisangiogenic vasculature. Thus, the present invention also providespeptides that home to nontumor angiogenic vasculature. Furthermore,these results indicate that tumor vasculature expressing targetmolecules that are not substantially expressed by other kinds ofangiogenic vasculature. Thus, the present invention provides a means toidentify target molecules expressed specifically by angiogenicvasculature present in a tumor, as well as for target moleculesexpressed by angiogenic vasculature not associated with a tumor. Methodsas disclosed herein can be used to distinguish such homing peptides andto isolate the various target molecules.

As an alternative to using a tumor sample to obtain the target molecule,extracts of cultured tumor cells or endothelial cells, depending onwhich cell type expresses the target molecule, can be used as thestarting material in order to enhance the concentration of the targetmolecule in the sample. It is recognized, however, that thecharacteristics of such cells can change upon adaptation to tissueculture. Thus, care must be exercised if such a preselection step isattempted. The presence of the target molecule can be established, forexample, by using phage binding and cell attachment assays (see, forexample, Barry et al., supra, 1996).

A cell line expressing a particular target molecule can be identifiedand surface iodination of the cells can be used to label the targetmolecule. The cells then can be extracted, for example, withoctylglucoside and the extract can be fractionated by affinitychromatography using a tumor homing peptide (see Tables 1 and 2) coupledto a matrix such as SEPHAROSE (see Hermanson, supra, 1996). The purifiedtarget molecule can be microsequenced and antibodies can be prepared. Ifdesired, oligonucleotide probes can be prepared and used to isolate cDNAclones encoding the target molecule. Alternatively, an anti-targetmolecule antibody can be used to isolate a cDNA clone from an expressionlibrary (see Argraves et al., J. Cell Biol. 105:1183-1190 (1987), whichis incorporated herein by reference).

As an alternative to isolating the target molecule, a nucleic acidencoding the target molecule can be isolated using a mammalian cellexpression cloning system such as the COS cell system. An appropriatelibrary can be prepared, for example, using mRNA from primary tumorcells. The nucleic acids can be cloned into the pcDNAIII vector(Invitrogen), for example. Cells expressing a cDNA for the targetmolecule can be selected by binding to the tumor homing peptide.Purified phage can be used as the carrier of the peptide and can beattached to magnetic beads coated, for example, with anti-M13 antibodies(Pharmacia Biotech; Piscataway N.J.). Cells that bind to the peptidecoating can be recovered using a magnet and the plasmids can beisolated. The recovered plasmid preparations then can be divided intopools and examined in COS cell transfections. The procedure can berepeated until single plasmids are obtained that can confer upon the COScells the ability to bind the tumor homing peptide.

The following examples are intended to illustrate but not limit thepresent invention.

Example I In Vivo Panning

This example demonstrates methods for preparing a phage library andscreening the library using in vivo panning to identify phage expressingpeptides that home to a tumor.

A. Preparation of Phage Libraries:

Phage display libraries were constructed using the fuse 5 vector asdescribed by Koivunen et al. (supra, 1995; Koivunen et al., supra,1994b). Libraries encoding peptides designated CX₅C (SEQ ID NO: 9), CX₆C(SEQ ID NO: 10), CX₇C (SEQ ID NO: 11) and CX₃CX₃CX₃C (SEQ ID NO: 12)were prepared, where “C” indicates cysteine and “X_(N)” indicates thegiven number of individually selected amino acids. These libraries candisplay cyclic peptides when at least two cysteine residues are presentin the peptide. In addition, a library that did not contain definedcysteine residues also was constructed. Such a library results in theproduction primarily of linear peptides, although cyclic peptides alsocan occur due to random probability.

A biased library based on the sequence CXXXNGRXX (SEQ ID NO: 13) alsowas constructed. Furthermore, in some cases the CXXXNGRXX (SEQ ID NO:13) library was further biased by in the incorporation of cysteineresidues flanking the NGR sequence, i.e., CXXCNGRCX (SEQ ID NO: 14; seeTable 1).

The libraries containing the defined cysteine residues were generatedusing oligonucleotides constructed such that “C” was encoded by thecodon TGT and “X_(N)” was encoded by NNK, where “N” is equal molarmixtures of A, C, G and T, and where “K” is equal molar mixtures of Gand T. Thus, the peptide represented by CX₅C (SEQ ID NO: 9) can berepresented by an oligonucleotide having the sequence TGT(NNK)₅TGT (SEQID NO: 14). Oligonucleotides were made double stranded by 3 cycles ofPCR amplification, purified and ligated to the nucleic acid encoding thegene III protein in the fuse 5 vector such that, upon expression, thepeptide is present as a fusion protein at the N-terminus of the gene IIIprotein.

The vectors were transfected by electroporation into MC1061 cells.Bacteria were cultured for 24 hr in the presence of 20 μg/mltetracycline, then phage were collected from the supernatant byprecipitation twice using polyethylene glycol. Each library containedabout 5×10⁹ to 5×10¹⁴ transducing units (TU; individual recombinantphage).

B. In Vivo Panning of Phage:

Tumors were transplanted into mice as described in Examples II and III,below. A mixture of phage libraries containing 1×10⁹ to 1×10¹⁴ TU wasdiluted in 200 μl DMEM and injected into the tail vein of anesthetizedmice (AVERTIN (0.015 ml/g); see U.S. Pat. No. 5,622,699; Pasqualini andRuoslahti, supra, 1996). After 1-4 minutes, mice were snap frozen inliquid nitrogen. To recover the phage, carcasses were partially thawedat room temperature for 1 hr, tumors and control organs were collectedand weighed, then were ground in 1 ml DMEM-PI (DMEM containing proteaseinhibitors (PI); phenylmethylsulfonyl fluoride (PMSF; 1 mM), aprotinin(20 μg/ml), leupeptin (1 μg/ml)).

Alternatively, following introduction of a library into a mouse,circulation of the library is terminated by perfusion through the heart.Briefly, mice were anesthetized with AVERTIN, then the heart was exposedand a 0.4 mm needle connected through a 0.5 mm cannula to a 10 ccsyringe was inserted into the left ventricle. An incision was made onthe right atrium and 5 to 10 ml of DMEM was slowly administered,perfusing the whole body over about a 5 to 10 min period. Efficiency ofthe perfusion was monitored directly by histologic analysis.

Tumor and organ samples were washed 3 times with ice cold DMEM-PIcontaining 1% bovine serum albumin (BSA); then directly incubated with 1ml K91-kan bacteria for 1 hr. Ten ml NZY medium containing 0.2 μg/mltetracycline (NZY/tet) was added to the bacterial culture, the mixturewas incubated in a 37° C. shaker for 1 hr, then 10 μl or 100 μl aliquotswere plated in agar plates containing 12.5 μg/ml tetracycline(tet/agar).

Individual colonies containing phage recovered from a tumor were grownfor 16 hr in 5 ml NZY/tet. The bacterial cultures obtained from theindividual colonies were pooled and the phage were purified andre-injected into mice as described above for a second round of in vivopanning. In general, a third round of panning also was performed. PhageDNA was purified from individual bacterial colonies obtained from thefinal round of in vivo panning and the DNA sequences encoding thepeptides expressed by selected phage were determined (see Koivunen etal., supra, 1994b).

Example II Identification of Tumor Homing Peptides by In Vivo PanningAgainst a Breast Tumor

This example demonstrates that in vivo panning can be performed againsta breast tumor to identify tumor homing peptides that home to varioustumors.

Human 435 breast carcinoma cells (Price et al., Cancer Res. 50:717-721(1990)) were inoculated into the mammary fat pad of nude mice. When thetumors attained a diameter of about 1 cm, either a phage targetingexperiment was performed, in which phage expressing a specific peptidewere administered to the tumor bearing mouse, or in vivo panning wasperformed.

The breast tumor bearing mice were injected with 1×10⁹ phage expressinga library of CX₃CX₃CX₃C (SEQ ID NO: 12) peptides, where X₃ indicatesthree groups of independently selected, random amino acids. The phagewere allowed to circulate for 4 min, then the mice were anesthetized,snap frozen in liquid nitrogen while under anesthesia, and the tumor wasremoved. Phage were isolated from the tumor and subjected to twoadditional rounds of in vivo panning.

Following the third round of panning, phage were quantitated and thepeptide sequences expressed by the cloned phage were determined. Thecloned phage expressed various different peptides, including those shownin Table 1. Similarly, CX₇C (SEQ ID NO: 11) and CX₅C (SEQ ID NO: 9)libraries were screened and breast tumor homing peptides were identified(Table 1). These results demonstrate that in vivo panning against abreast tumor can identify tumor homing molecules.

Example III In Vivo Targeting of a Phage Expressing an an RGD Peptide toa Tumor

Human 435 breast carcinoma cells were inoculated into the mammary fatpad of nude mice. When the tumors attained a diameter of about 1 cm,phage expressing a specific RGD-containing peptide were administered tothe tumor bearing mouse. Similar results

TABLE 1 PEPTIDES FROM PHAGE RECOVERED FROM HUMAN BREAST CANCERCGRECPRLCQSSC  (2*) CNGRCVSGCAGRC   (3) CGEACGGQCALPC  (20) IWSGYGVYW (21) PSCAYMCIT  (22) WESLYFPRE  (23) SKVLYYNWE  (24) CGLMCQGACFDVC (25) CERACRNLCREGC  (26) CPRGCLAVCVSQC  (27) CKVCNGRCCG  (28)CEMCNGRCMG  (29) CPLCNGRCAL  (30) CPTCNGRCVR  (31) CGVCNGRCGL  (32)CEQCNGRCGQ  (33) CRNCNGRCEG  (34) CVLCNGRCWS  (35) CVTCNGRCRV  (36)CTECNGRCQL  (37) CRTCNGRCLE  (38) CETCNGRCVG  (39) CAVCNGRCGF  (40)CRDLNGRKVM  (41) CSCCNGRCGD  (42) CWGCNGRCRM  (43) CPLCNGRCAR  (44)CKSCNGRCLA  (45) CVPCNGRCHE  (46) CQSCNGRCVR  (47) CRTCNGRCQV  (48)CVQCNGRCAL  (49) CRCCNGRCSP  (50) CASNIQGRVVL  (51) CGRCNGRCLL  (52)CWLCNGRCGR  (53) CSKCNGRCGH  (54) CVWCNGRCGL  (55) CIRCNGRCSV  (56)CGECNGRCVE  (57) CEGVNGRRLR  (58) CLSCNGRCPS  (59) CEVCNGRCAL  (60)CGSLVRC   (5) GRSQMQI  (61) HHTRFVS  (62) SKGLRHR  (63) VASVSVA  (64)WRVIJIAF  (65) KMGPKVW  (66) IFSGSRE  (67) SPGSWTW  (68) NPRWFWD  (69)GRWYKWA  (70) IKARASP  (71) SGWCYRC  (72) ALVGLMR  (73) LWAEMTG  (74)CWSGVDC  (75) DTLRLRI  (76) SKSSGVS  (77) IVADYQR  (78) VWRTGHL  (79)VVDRFPD  (80) LSMFTRP  (81) GLPVKWS  (82) IMYPGWL  (83) CVMVRDGDC  (84)CVRIRPC  (85) CQLAAVC  (86) CGVGSSC  (87) CVSGPRC  (88) CGLSDSC  (89)CGEGHPC  (90) CYTADPC  (91) CELSLISKC  (92) CPEHRSLVC  (93) CLVVHEAAC (94) CYVELHC  (95) CWRKFYC  (96) CFWPNRC  (97) CYSYFLAC  (98) CPRGSRC (99) CRLGIAC (100) CDDSWKC (101) CAQLLQVSC (102) CYPADPC (103) CKALSQAC(104) CTDYVRC (105) CGETMRC (106) *-numbers in parentheses indicate SEQID NO:.to those discussed below also were obtained with nude mice bearingtumors formed by implantation of human melanoma C8161 cells or byimplantation of mouse B16 melanoma cells.

1×10⁹ phage expressing the RGD-containing peptide, CDCRGDCFC (SEQ ID NO:1; see, Koivunen et al., supra, 1995) or control (insertless) phage wereinjected intravenously (iv) into the mice and allowed to circulate for 4min. The mice then were snap frozen or perfused through the heart whileunder anesthesia, and various organs, including tumor, brain and kidney,were removed and the phage present in the organs was quantitated (seeU.S. Pat. No. 5,622,699; Pasqualini and Ruoslahti, supra, 1996).

Approximately 2-3 times more phage expressing the CDCRGDCFC (SEQ IDNO: 1) peptide were detected in the breast tumor as compared to brainand kidney, indicating the CDCRGDCFC (SEQ ID NO: 1; RGD phage) peptideresulted in selective homing of the phage to the breast tumor. In aparallel study, unselected phage, which express various, diversepeptides, were injected into tumor-bearing mice and various organs wereexamined for the presence of phage. Far more phage were present inkidney and, to a lesser extent, brain, as compared to the tumor. Thus,the 80-fold more RGD-expressing phage than unselected phage concentratedin the tumor. These results indicate that phage expressing theRGD-containing peptide home to a tumor, possibly due to the expressionof the α_(v)β₃ integrin on blood vessels forming in the tumor.

Specificity of the breast tumor homing peptide was demonstrated bycompetition experiments, in which coinjection of 500 μg free peptide,ACDCRGDCFCG (SEQ ID NO: 16; see Pasqualini et al., supra, 1997) with thephage expressing the tumor homing peptide reduced the amount of phage inthe tumor by about tenfold, whereas coinjection with the inactivecontrol peptide, GRGESP (SEQ ID NO: 17) essentially had no effect. Theseresults demonstrate that phage displaying a peptide that can bind to anintegrin expressed on angiogenic vasculature can selectively home invivo to an organ or tissue such as a tumor containing such vasculature.

Example IV Immunohistologic Analysis of Tumor Homing Peptides

This example provides a method of identifying the localization of tumorhoming molecules by immunohistologic examination.

Localization of phage expressing a tumor homing peptide was identifiedby immunochemical methods in histologic sections obtained either 5 minor 24 hr after administration of phage expressing a tumor homing peptide(“peptide-phage”) to a tumor bearing mouse (FIG. 1). For samplesobtained 5 min following administration of the peptide-phage, mice wereperfused with DMEM and various organs, including the tumor, were removedand fixed in Bouin's solution. For samples obtained at 24 hr, nopeptide-phage remains in the circulation and, therefore, perfusion wasnot required. Histologic sections were prepared and reacted withanti-M13 (phage) antibodies (Pharmacia Biotech; see U.S. Pat. No.5,622,699; Pasqualini and Ruoslahti, supra, 1996). Visualization of thebound anti-M13 antibody was performed using a peroxidase-conjugatedsecond antibody (Sigma; St. Louis Mo.) according to the manufacturer'sinstructions.

As discussed in Example III, phage expressing the tumor homing peptide,CDCRGDCFC (SEQ ID NO: 1; “RGD phage), were administered intravenously tomice bearing the breast tumor. In addition, the RGD phage wereadministered to mice bearing a mouse melanoma or a human Kaposi'ssarcoma. Circulation of the phage was terminated and mice weresacrificed as described above and samples of the tumor and of skinadjacent to the tumor, brain, kidney, lung and liver were collected.Immunohistochemical staining for the phage showed accumulation of theRGD phage in the blood vessels present in the breast tumor as well as inthe melanoma and the Kaposi's sarcoma, whereas little or no staining wasobserved in the control organs.

Similar experiments were performed using phage expressing the tumorhoming peptide, CNGRCVSGCAGRC (SEQ ID NO: 3; “NGR phage”), which wasidentified by in vivo panning against a tumor formed by the MDA-MB-435breast carcinoma. In these experiments, NGR phage or control phage,which do not express a peptide, were administered to mice bearing tumorsformed by the MDA-MB-435 breast carcinoma or by a human SLK Kaposi'ssarcoma xenograft, then the mice were sacrificed as described above andtumors were collected as well as control organs, including brain, lymphnode, kidney, pancreas, uterus, mammary fat pad, lung, intestine, skin,skeletal muscle, heart and epithelium of the renal calices, bladder andureter (see FIG. 1). Histological samples were prepared and examined byimmunostaining as described above.

In samples obtained from mice sacrificed 4 min after administration ofthe NGR phage, immunostaining of the vasculature of both the breasttumor (FIG. 1E) and the Kaposi's sarcoma (FIG. 1H) was observed. Verylittle or no staining was observed in the endothelium of the thesetumors in mice administered an insertless control phage (FIGS. 1G and1J, respectively). In the samples obtained from mice sacrificed 24 hrafter administration of the NGR phage, staining of the tumor samplesappeared to have spread outside of the vessels, into the breast tumorparenchyma (FIGS. 1B and 1F) and the Kaposi's sarcoma parenchyma.(FIGS.1D and 1I). Again, little or no staining was observed in samplesprepared from these tumors in mice administered the insertless controlphage (Figures A and C, respectively). In addition, little or nostaining was observed in various control organs in mice administered theNGR phage (FIGS. 1K to 1V).

In other experiments, similar results were obtained followingadministration of phage expressing the NGR tumor homing peptides, NGRAHA(SEQ ID NO: 6) or CVLNGRMEC (SEQ ID NO: 7), to tumor bearing mice. Also,as discussed below, similar results were obtained using phage expressingthe GSL tumor homing peptide, CLSGSLSC (SEQ ID NO: 4), which wasidentified by in vivo panning of a melanoma (see Example V, below).

These results demonstrate that tumor homing peptides selectively home totumors, particularly to the vasculature in the tumors and that tumorhoming peptides identified, for example, by in vivo panning against abreast carcinoma also selectively home to other tumors, includingKaposi's sarcoma and melanoma. In addition, these results demonstratethat immunohistochemical analysis provides a convenient assay foridentifying the localization of phage expressing tumor homing peptides.

Example V Identification of Tumor Homing Peptides by In Vivo PanningAgainst a Melanoma Tumor

The general applicability of the in vivo panning method to identifytumor homing peptides was examined by performing in vivo panning againstan implanted mouse melanoma tumor.

Mice bearing a melanoma were produced by implantation of B16B15b mousemelanoma cells, which produce highly vascularized tumors. B16B15b mousemelanoma cells were injected subcutaneously into the mammary fat pad ofnude mice (2 months old) and tumors were allowed to grow until thediameter was about 1 cm. In vivo panning was performed as disclosedabove. Approximately 1×10¹² transducing units of phage expressing theCX₅C (SEQ ID NO: 9), CX₆C (SEQ ID NO: 10) or CX₇C (SEQ ID NO: 11)library were injected, iv, and allowed to circulate for 4 min. Mice thenwere snap frozen in liquid nitrogen or perfused through the heart whileunder anesthesia, tumor tissue and brain (control organ) were removed,and phage were isolated as described above. Three rounds of in vivopanning were performed.

The amino acid sequences were determined for the inserts in 89 clonedphage recovered from the B16B15b tumors. The peptides expressed by thesephage were represented by two predominant sequences, CLSGSLSC (SEQ IDNO: 4; 52% of the clones sequenced) and WGTGLC (SEQ ID NO: 18; 25% ofthe clones; see Table 2). Reinfection of phage expressing one of theselected peptides resulted in approximately three-fold enrichment ofphage homing to the tumor relative to brain.

TABLE 2 PEPTIDES FROM PHAGE RECOVERED FROM MOUSE B16B15b MELANOMACLSGSLSC  (4*) GICKDDWCQ (107) TSCDPSLCE (108) KGCGTRQCW (109) YRCREVLCQ(110) CWGTGLC (111) WSCADRTCM (112) AGCRLKSCA (113) SRCKTGLCQ (114)PICEVSRCW (115) WTCRASWCS (116) GRCLLMQCR (117) TECDMSRCM (118)ARCRVDPCV (119) CIEGVLGGC (120) CSVANSC (121) CSSTMRC (122) SIDSTTF(123) GPSRVGG (124) WWSGLEA (125) LGTDVRQ (126) LVGVRLL (127) GRPGDIW(128) TVWNPVG (129) GLLLVVP (130) FAATSAE (131) WCCRQFN (132) VGFGKAL(133) DSSLRLP (134) KLWCAMS (135) SLVSFLG (136) GSFAFLV (137) IASVRWA(138) TWGHLRA (139) QYREGLV (140) QSADRSV (141) YMFWTSR (142) LVRRWYL(143) TARGSSR (144) TTREKNL (145) PKWLLFS (146) LRTNVVH (147) AVMGLAA(148) VRNSLRN (149) *- numbers in parentheses indicate SEQ ID NO:.

Localization of the phage expressing a tumor homing peptide in the mouseorgans also was examined by immunohistochemical staining of the tumorand various other tissues (see Example IV). In these experiments, 1×10⁹pfu of a control (insertless) phage or a phage expressing the tumorhoming peptide, CLSGSLSC (SEQ ID NO: 4), were injected, iv, into tumorbearing mice and allowed to circulate for 4 min.

Immunostaining was evident in the melanoma obtained from a mouseinjected with phage expressing the CLSGSLSC (SEQ ID NO: 4) tumor homingpeptide. Staining of the melanoma generally was localized to the bloodvessels within the tumor, although some staining also was present in thetumor parenchyma. Essentially no staining was observed in a tumorobtained from a mouse injected with the insertless control phage or inskin or in kidney samples obtained from mice injected with either phage.However, immunostaining was detected in the liver sinusoids and inspleen, indicating that phage can be trapped nonspecifically in organscontaining RES.

Using similar methods, in vivo panning was performed in mice bearing aSLK human Kaposi's sarcoma. Tumor homing peptides were identified andare disclosed in Table 3. Together, these results demonstrate that thein vivo panning method is a generally applicable method for screening aphage library to identify phage expressing tumor homing peptides.

Example VI Preparation and Characterization of Tumor HomingPeptide/Doxorubicin Conjugates

This example provides methods for conjugating a moiety such as thechemotherapeutic agent, doxorubicin, to a tumor homing peptide and forcharacterizing the conjugation reaction.

The peptides CDCRGDCFC (SEQ ID NO: 1; Koivunen et al., supra, 1995;Pasqualini et al., supra, 1997), CNGRC (SEQ ID NO: 8), CGSLVRC (SEQ IDNO: 5) and GACVFSIAHECGA (SEQ ID NO: 19) were synthesized, cyclizedunder high-dilution and purified, to homogeneity by HPLC. Conjugation ofthe peptides to doxorubicin (Aldrich; Milwaukee Wis.) was performedusing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC;Sigma; St. Louis Mo.) and N-hydroxysuccinimide (NHS;

TABLE 3 PEPTIDES FROM PHAGE RECOVERED FROM HUMAN KAPOSI'S SARCOMATDCTPSRCT (150*) SWCQFEKCL (151) VPCRFKQCW (152) CTAMRNTDC (153)CRESLKNC (154) CMEMGVKC (155) VTCRSLMCQ (156) CNNVGSYC (157) CGTRVDHC(158) CISLDRSC (159) CAMVSMED (160) CYLGVSNC (161) CYLVNVDC (162)CIRSAVSC (163) LVCLPPSCE (164) RHCFSQWCS (165) FYCPGVGCR (166) ISCAVDACL(167) EACEMAGCL (168) PRCESQLCP (169) RSCIKHQCP (170) QWCSRRWCT (171)MFCRMRSCD (172) GICKDLWCQ (173) NACESAICG (174) APCGLLACI (175)NRCRGVSCT (176) FPCEGKKCL (177) ADCRQKPCL (178) FGCVMASCR (179)AGCINGLCG (180) RSCAEPWCY (181) DTCRALRCN (182) KGCGTRQCW (109)GRCVDGGCT (183) YRCIARECE (184) KRCSSSLCA (185) ICLLAHCA (186) QACPMLLCM(187) LDCLSELCS (188) AGCRVESC (189) HTCLVALCA (190) IYCPGQECE (191)RLCSLYGCV (192) RKCEVPGCQ (193) EDCTSRFCS (194) LECVVDSCR (195)EICVDGLCV (196) RWCREKSCW (197) FRCLERVCT (198) RPCGDQACE (199)CNKTDGDEGVTC  (15) *- numbers in parentheses indicate SEQ ID NO:.Sigma) as described (Bauminger and Wilchek, supra, 1980; Harlow andLane, supra, 1988; Hurwitz et al., supra, 1975). Unreacted doxorubicinand peptide were removed from the doxorubicin/peptide conjugates bySEPHADEX G25 column chromatography using phosphate buffered saline. Theconjugates were lyophilized for storage and were resuspended in sterilewater prior to use.

HPLC, capillary electrophoresis and NMR analyses were performed tocharacterize the conjugates. HPLC-fluorescence was performed using anINTERSIL ODS-2 column (4.6×150 mm) and a mobile phase composed of 0.08%triethanolamine/0.02% phosphoric acid (85%)/27% acetonitrile at 1ml/min. Fluorescence detection was performed with excitation at 490 nmand emission at 560 nm wavelength and the retention time (RT) and thearea under the curves (AUC) for doxorubicin (dox) and for the majorpeaks was determined. Each of the conjugates has a unique retentiontime, depending on the peptide, as follows: dox/CDCRGDCFC (SEQ ID NO:1), RT 7.4 min, AUC 26%; dox/CNGRC (SEQ ID-NO: 8), RT 4.7 min, AUC 56%;and dox/GACVFSIAHECGA (SEQ ID NO: 19), RT 7.7 min, AUC 43%. Incomparison, the retention time of doxorubicin is 10.6 min and, in thevarious reactions, the AUC was about 5%.

Capillary electrophoresis (CE; Liu et al., supra, 1996) was performed inuncoated fused-silica capillaries with 75 μm internal diameter and aneffective separation length of 50 cm. The CE detection system wasequipped with an UV absorbance detector and an argon laser emitting at488 nm. The laser beam is transmitted via a fiber optic cable to thedetector and illuminates the capillary window and the fluorescencesignal is collected through an emission filter. Conjugation ofdoxorubicin to the peptides changed the electrophoretic characteristicsof each of the conjugates, indicating that this method can be used as afast screening method to identify progress of the conjugation reaction.

One dimensional NMR analysis of the doxorubicin/CNGRC conjugate revealedno evidence of resonances arising from free doxorubicin. Two dimensionNMR analysis can allow a determination of the precise molecularstructure of the doxorubicin-peptide species. These results demonstratethat a moiety such as the cancer chemotherapeutic agent, doxorubicin,can be efficiently linked to tumor homing peptides of the invention toproduce doxorubicin/tumor homing peptide conjugates.

Example VII Tumor Therapy Using Doxorubicin/Tumor Homing PeptideConjugates

This example demonstrates that doxorubicin/tumor homing peptideconjugates provide a therapeutic advantage over the use of doxorubicin,alone, for treating tumors.

Doxorubicin concentration of the conjugates (see Example V) wasdetermined by measuring the optical absorbance of the solution at 490 nmin a standard spectrophotometer; this wavelength detects only thedoxorubicin, not the peptides. A calibration curve for doxorubicin wasgenerated and used to calculate the concentration prior to use.Conjugation of doxorubicin to the various peptides did not affect thiscurve. This procedure ensures that each of the administered conjugatescontained the same amounts of doxorubicin equivalent.

In addition, the viability of tumor cells obtained from tumors of micetreated with a tumor homing peptide/doxorubicin conjugate was comparedto that of tumors from mice treated with free doxorubicin. In theseexperiments, breast tumor bearing mice were size matched with regard tothe tumors, then treated intravenously with 30 μg equivalent ofdoxorubicin/CDCRGDCFC (SEQ ID NO: 1) or of free doxorubicin. Five daysafter treatment, the mice were euthanized and the tumors were removed.The tumor pairs were weighed and ground and the cell suspensions wereplated (2 g tumor tissue per 150 mm plate).

Cell numbers were determined at 24 hours and 7 days after plating.Viability of tumor cells from the tumors of mice receiving thedoxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate was about 3 fold lessthan cells from tumors of mice treated with the free doxorubicin. Theseresults demonstrate that administration to a tumor bearing mouse of aconjugate comprising a chemotherapeutic agent linked to a tumor homingmolecule is more efficacious than administration of the agent, alone, inreducing the viability of tumor cells.

A. In Vitro Characterization of Cytotoxicity:

MDA-MB-435 human breast carcinoma cells were plated at 1×10⁵ cells/wellin 96 well plates. Cells were incubated with increasing amounts ofdoxorubicin, the doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate, or thedoxorubicin/GACVFSIAHECGA (SEQ ID NO: 19; control) conjugate (0.1 to 10μg/well of doxorubicin-equivalent) for either 30 min or overnight.Following incubation, the agents were removed by extensive washing withPBS, then fresh medium added and incubation was continued. The number ofsurviving cells was determined at 24 hours with crystal violet staining(see Koivunen et al., supra, 1994).

In cells exposed to free doxorubicin, the doxorubicin/CDCRGDCFC (SEQ IDNO: 1) conjugate, or the doxorubicin/GACVFSIAHECGA (SEQ ID NO: 19) for30 min, cell death was present only in the cultures treated with thedoxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate. However, if the agentswere not removed after 30 min, the cells were killed by all of thetreatments after 24 hr. These results indicate that enhanced cellularuptake occurs for the doxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate.

B. In Vivo Characterization of Doxorubicin/Tumor Homing PeptideConjugates:

Female 2-month old Balb c nu/nu mice (Harlan Sprague Dawley; San DiegoCalif.) were used for these studies and were cared for according to theBurnham Institute animal facility guidelines. MDA-MB-435 breastcarcinoma cells (Price et al., Cancer Res. 50:717-721 (1993)) wereinjected in the mammary fat pad of the nude mice and tumor growth wasmonitored (Pasqualini et al., supra, 1997). Tumors were allowed to growto a size of about 1 cm³ (about 5% of the mouse's body weight) beforestarting the treatment experiments, except for the toxicity experimentsas discussed below.

Weekly doxorubicin/peptide conjugate or control treatments (5μg/mouse/week of doxorubicin-equivalent) were administeredintravenously. In some experiments, as indicated, a dose of 30 μg/mousewas administered every 3 weeks. Treatment with doxorubicin, alone, isreferred to as “dox control” and treatment with doxorubicin conjugatedto the non-tumor homing control peptide, GACVFSIAHECGA (SEQ ID NO: 19),is referred to as “conjugate control.” The results obtained in the doxcontrol groups as compared to the conjugate control groups were notsignificantly different. As an additional control, in some experimentsthe tumor homing peptide was mixed with doxorubicin, without linking,and the mixture was administered to tumor bearing mice. Such treatmentproduced results that were not statistically different from thoseobtained with the above described dox controls.

Mice were anesthetized with a tribromoethanol based anesthetic mixture(AVERTIN; Papaioannou and Fox, Lab. Anim. 43:189-192 (1993)) before eachtreatment. Anesthetization facilitated the tail vein injections (finalvolume, 200 μl) and allowed precise serial three dimensional tumor sizemeasurements. Tumor volume calculations were based on the equation forthe volume of an ovaloid: V=4/3(nabc), where a, b, and c are 1/2 of themeasured diameters in each of the three dimensions.

At necropsy, MDA-MB-435 tumor-bearing mice treated with thedoxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate had significantly smallertumors (t test, p=0.02), less spread to regional lymph nodes (t test,p<0.0001), a lower incidence of pulmonary metastasis and fewermetastatic lesions (t test, p<0.0001) than the dox control treated mice.All of the mice treated with the doxorubicin/CDCRGDCFC (SEQ ID NO: 1)conjugate survived beyond the time when the dox control and conjugatecontrol mice had died (Log-Rank test, p<0.0001; Wilcoxon test,p=0.0007). Essentially the same results were obtained in five separateexperiments. These results indicate that a doxorubicin/tumor homingpeptide provides a therapeutic advantage over doxorubicin, alone, inreducing the growth of a primary tumor and preventing metastasis of thetumor.

Gross and histopathologic examination was performed on the mice. Many ofthe tumors in the mice treated with 5 μg doxorubicin equivalent ofdoxorubicin/CDCRGDCFC (SEQ ID NO: 1) presented marked skin ulcerationand tumor necrosis, whereas no such signs were observed in dox controlgroup or conjugate control group. Histopathological analysis disclosed apronounced destruction of the vasculature in the tumors treated withdoxorubicin/CDCRGDCFC (SEQ ID NO: 1) conjugate (see FIGS. 2D to 2F) ascompared to the dox control group (FIGS. 2A to 2C).

In a dose escalation experiment, tumor bearing mice were treated withthe doxorubicin/CDCRGDCFC (SEQ ID NO: 1) at 30 μg/mouse every threeweeks for three cycles and were observed, without further treatment, foran extended period of time. The doxorubicin/CDCRGDCFC (SEQ ID NO: 1)treated mice all remained alive more than 6 months after the dox controland conjugate control mice had died. The results indicate that treatmentwith a doxorubicin/tumor homing peptide conjugate can have a curativeeffect.

Acute toxicity studies also were performed. In these experiments, micebearing extremely large tumors (about 25% of body weight) were treatedwith 200 μg/mouse doxorubicin or doxorubicin/CDCRGDCFC (SEQ ID NO: 1)conjugate. All of the mice treated with the doxorubicin/CDCRGDCFC (SEQID NO: 1) conjugate survived for longer than one week, whereas all ofthe dox control mice had died within 48 hr of treatment. These resultssuggest that accumulation of the doxorubicin/CDCRGDCFC (SEQ ID NO: 1)conjugate in the large tumors reduced the circulating level of thecorijugatei3 doxorubicin, thus reducing its toxicity.

Similar results were obtained using the doxorubicin/CNGRC (SEQ ID NO: 8)conjugate. In each of three series of experiments, tumors in the micetreated with doxorubicin/CNGRC (SEQ ID NO: 8) were significantly smallerthan tumor is the dox control and conjugate control groups. Treatmentwith the doxorubicin/CNGRC (SEQ ID NO: 8) conjugate almost completelysuppressed tumor growth, whereas free doxorubicin and doxorubicinconjugated to the control peptide had essentially no effect on tumorgrowth relative to treatment with the vehicle, alone. A marked effect onsurvival also was observed and some of the doxorubicin/CNGRC (SEQ ID NO:8) treated animals survived for extended periods of time (Log-Rank test,p=0.0064; Wilcoxon test, p=0.0343). In addition, the doxorubicin/CNGRC(SEQ ID NO: 8) conjugate was less toxic than free doxorubicin. Theseresults confirm that conjugates comprising a chemotherapeutic agent anda tumor homing molecule provide a therapeutic advantage in treatingcancer.

Although the invention has been described with reference to thedisclosed examples, it should be understood that various modificationscan be made without departing from the spirit of the invention.Accordingly, the invention is limited only by the following claims.

1-15. (canceled)
 15. A conjugate, comprising a tumor homing moleculelinked to a moiety, said tumor homing molecule obtained by in vivopanning, comprising the steps of: a) administering to a first subjecthaving a tumor a library of diverse molecules; b) collecting a sample ofthe tumor; c) identifying a molecule that homes to said tumor; d)collecting a sample of normal tissue corresponding to said tumor; and e)determining that said molecule that homes to said tumor is not presentin said normal tissue, thereby obtaining said tumor homing molecule,provided said tumor homing molecule is not an antibody.
 16. Theconjugate of claim 15, wherein said molecule is a nucleic acid molecule.17. The conjugate of claim 15, wherein said molecule is apeptidomimetic. 18-28. (canceled)
 29. A tumor homing peptide identifiedby in vivo panning, comprising the steps of: a) administering to a firstsubject having a tumor a library of diverse peptides; b) collecting asample of the tumor; c) identifying a peptide that homes to said tumor;d) collecting a sample of normal tissue corresponding to said tumor; ande) determining that said peptide that homes to said tumor is not presentin said normal tissue, thereby identifying said peptide as a tumorhoming peptide, provided said peptide is not an antibody.
 30. The tumorhoming peptide of claim 29, wherein said sample of normal tissuecorresponding to said tumor is collected from said first subject. 31.The tumor homing peptide of claim 29, wherein said sample of normaltissue corresponding to said tumor is collected from a second subject.32. The tumor homing peptide of claim 29, wherein said tumor is a breasttumor.
 33. The tumor homing peptide of claim 29, wherein said tumor is amelanoma.
 34. The tumor homing peptide of claim 29, wherein said tumoris a Kaposi's sarcoma.
 35. A tumor homing peptide selected from thegroup consisting of CLSGSLSC (SEQ ID NO: 4) and CGSLVRC (SEQ ID NO: 5).36-44. (canceled)
 45. A molecule, which specifically binds to the targetmolecule of claim
 43. 46. A molecule, which competitively inhibits thebinding of the target molecule of claim 43 to the tumor homing molecule.47. The molecule of claim 46, which is a peptide.
 48. The peptide ofclaim 47, which contains the amino acid sequence NGR.
 49. The peptide ofclaim 48, which has the amino acid sequence CNGRCVSGCAGRC (SEQ ID NO:3).
 50. The peptide of claim 47, which contains the amino acid sequenceGSL.
 51. The peptide of claim 50, which has the amino acid sequenceCGSLVRC (SEQ ID NO: 5).
 52. A target molecule, which is expressed intumor vasculature, wherein said target molecule binds CNGRC (SEQ ID NO:8) with a higher affinity than said target molecule binds CDCRGDCFC (SEQID NO: 1).
 53. An antibody that specifically binds the target moleculeof claim
 43. 54. The antibody of claim 53, which is a monoclonalantibody. 55-72. (canceled)